abstract: # Background
  .
  The clinical learning environment is a complex social entity that influences student learning outcomes in the clinical setting. Students can experience the clinical learning environment as being both facilitative and obstructive to their learning. The clinical environment may be a source of stress, creating feelings of fear and anxiety which in turn affect the students' responses to learning. Equally, the environment can enhance learning if experienced positively.
  .
  # Objectives
  .
  This study described pupil enrolled nurses' experiences of facilitative and obstructive factors in military and public health clinical learning settings.
  .
  # Method
  .
  Using a qualitative, contextual, exploratory descriptive design, three focus group interviews were conducted until data saturation was reached amongst pupil enrolled nurses in a military School of Nursing.
  .
  # Results
  .
  Data analysed provided evidence that acceptance by clinical staff and affordance of self-directed learning facilitated learning. Students felt safe to practise when they were supported by the clinical staff. They felt a sense of belonging when the staff showed an interest in and welcomed them. Learning was obstructed when students were met with condescending comments. Wearing of a military uniform in the public hospital and horizontal violence obstructed learning in the clinical learning environment.
  .
  # Conclusion
  .
  Students cannot have effective clinical preparation if the environment is not conducive to and supportive of clinical learning, The study shows that military nursing students experience unique challenges as they are trained in two professions that are hierarchical in nature. The students experienced both facilitating and obstructing factors to their learning during their clinical practice. Clinical staff should be made aware of factors which can impact on students' learning. Policies need to be developed for supporting students in the clinical learning environment.
author: Eucebious Lekalakala-Mokgele; Ernestine M. Caka**Correspondence to:** Eucebious Lekalakala-Mokgele **Email:**<firstname.lastname@example.com>**Postal address:** PO Box 72, Medunsa 0204, South Africa[^1]
date: 2015-03-31
references:
title: Facilitative and obstructive factors in the clinical learning environment: Experiences of pupil enrolled nurses

# Introduction

Military health nurse training differs from general nurse training in South Africa. Nurses are usually trained in private or public schools and colleges of nursing for a period of two years (for enrolled nurses) and four years (for comprehensive basic nursing) (South African Nursing Council 1975; 1993a; 1993b). The military in South Africa train their own enrolled nurses within the military schools of nursing for two years as pupil enrolled nurses (PEN), although under the same South African Nursing Council regulation 2175 for the training of regular enrolled nurses (South African Nursing Council 1993a). The military PEN commence with a six-month basic military training to prepare them as soldiers first before they embark on a two-year nursing programme, on completion of which they are registered as enrolled nurses.

Maginnis and Croxon (2007:218) insist that the clinical placement of students needs to prepare them adequately for practice in an environment that is constantly changing. Whilst nurses are generally trained in corresponding private and public clinical learning environments (CLEs), military nurses receive their own clinical learning experiences in the military health and public health institutions, for diverse clinical placement and to be exposed to different health care experiences that will equip them to nurse holistically (Purdie, Sheward & Gifford 2008:315).

This arrangement of placing military students in public health institutions was made because military hospitals were initially used only as a sickbay (a room for treating sick and injured soldiers) and for visiting the doctor on an outpatient basis. They therefore lacked diverse clinical exposure for their students. These students, including PEN, have to experience different challenges in two diverse CLEs. In the military health system only members of the military (various ranks) and their family members may be admitted as patients, whereas the public health sector accommodates civilian patients from all of the different spheres of South African society (Caka & Lekalakala-Mokgele 2013:2).

## Problem statement

The CLE constitutes a clinical setting which offers the students an opportunity to practise the necessary skills to enable them provide patient care (Cheraghi *et al*. 2008:26). The uniqueness of the CLE in the military health service means that the experiences of the PENs in the military are different from those in the public sector. Firstly, their workplace learning takes place in a military milieu where power ranks are more pronounced. It is where they may have to treat high-ranking personnel as their patients and have to also contend with high-ranking clinical staff who are often also trained in military nursing and military culture. Secondly, because of the limited number of clinical areas in the military health service, students are allocated to public hospitals where they take care of civilian patients under different conditions than exist in the military setting.

Literature reveals that there are both facilitative and obstructive factors to students' learning within this environment (Löfmark & Wikblad 2001:49). The researcher's personal experience of five years as an educator in the military health service, as well as her concern about the need to understand the dynamics that facilitate or obstruct learning, led to initiation of this project in order to investigate the experiences of PEN in the military health service. This has not been done previously in South Africa and it was not known how these PEN would describe their experiences in these two diverse clinical settings. The following research question was asked: 'How do military PEN experience their clinical learning environment?'

## Definition of key concepts

### Clinical learning environment

In this study, the CLE is a learning environment accredited by the South African Nursing Council where students acquire the knowledge and skills to manage both military patients of various ranks and their family members. It also includes the public health sector which accommodate civilian patients from all spheres of South African society.

### Military health setting

This is a formal setting for the development of various skills, including clinical skills, that will prepare PEN in times of war and defence of the country. PEN are trained to care for sick or injured soldiers and their families in the line of battle.

### Public health setting

A setting that mobilises local, State, national and international resources to ensure optimal conditions for people's health. It comprises preventive, curative and rehabilitative services (Basu, Jina & Naidoo 2008:7).

### Pupil enrolled nurses

This term refers to persons undergoing a two-year programme at an approved nursing school, who have complied with the prescribed conditions and have furnished the prescribed particulars as required by section 24 *x* of the *Nursing Act* 50 of 1978 (Republic of South Africa 1978).

## Literature review

Literature suggests that nursing students' clinical placement experiences are often fraught with longitudinal and multidimensional challenges (Clare *et al*. 2003). These challenges include a lack of support of students (from both the CLE staff and their facilitators), which can lead to missed opportunities in terms of teachable moments (Newton, Billet & Ockerby 2009). Reports suggest that nurses deal more with documentation than patient care – the feeling that there is a lack of interpersonal relationships between students, staff members and the patients is also documented (Pearcey & Draper 2008:595). The CLE is even recognised as a source of stress that creates feelings of fear and anxiety, which in turn affect the students' responses to learning (Chesser-Smyth 2005:320).

According to Levett-Jones and Lathlean (2009:348) students can experience the CLEs as belligerent if they are constantly criticised and judged harshly by clinical staff. For many students, clinical placements are typified by a feeling of alienation and a lack of belonging (Goh & Watt 2003:14; Newton *et al*. 2009:633). These behaviours can be obstructive to learning. According to Löfmark and Wikblad (2001:46), the CLE is obstructive to learning when there is no student–supervisor relationship and/or when supervisors make condescending comments, are irritated or not interested in students and do not allow students to take part in patient care activities.

Violence is another factor that is obstructive to learning. Carley and Mackway-Jones (2005:26) suggest that nursing profession is hierarchical and is embedded in the military system. This hierarchy results in student nurses experiencing 'horizontal violence' (Curtis, Bowen & Reid 2007:156), with junior members in the profession being bullied, ignored and disrespected.

Facilitative factors are considered to be those that promote learning. Literature has identified multiple such factors, amongst others a sense of belonging and acceptance. A number of affective consequences are said to influence the student's sense of belonging, including feeling safe, comfortable, satisfied and happy (Levett-Jones & Lathlean 2008:103). A sense of belonging is underpinned by a need for affirmation, friendship and being part of the group (Friedkin 2004:410). When students realise that they are not part of the collective, they may present with low self-esteem and unhappiness, which may result in a diminished learning process (Baumeister, Twenge & Nuss 2002:817). It is thus important to inculcate a sense of belonging that produces happy group members, because students who feel ostracised by their peers or those in authority will not function optimally (Tabane & Human-Vogel 2010:504).

According to Löfmark and Wikblad (2001:47), being allowed to take responsibility and to work independently, having opportunities to practise tasks and receive feedback, collaborating with staff and supervising others, gaining an overview of the setting and a sense of control, are all factors considered to facilitate learning in the CLE. Levett-Jones *et al*. (2007:211) assert that a feeling of acceptance, being valued and being involved with others are some of the attributes of belonging. The purpose of this article is to explore and describe PEN experiences of facilitative and obstructive factors in clinical learning in two diverse settings.

# Research methods and design

## Design

An explorative, descriptive, contextual qualitative research approach was used in this study to explore and describe the experiences of PEN in the military health CLE and within public health settings.

## Population and sample

There was a total of 30 PEN in the studied institution at the time of collecting data for this study, which included PEN in their second year of training. The second-year PEN have been in training for over a year and have been more exposed to the various clinical learning situations, including placements in public hospitals. Criteria for inclusion in this study were that participants should be registered as PEN in the Military Nursing College, must be in their second year of study and should have been allocated to the military health CLE and public hospitals. The final sample size, using the eligibility criteria, was determined by data saturation (Streubert & Carpenter 2011:90).

## Data collection method

Data were collected by means of focus group interviews. A total of 19 students participated in the study voluntarily. Three focus group sessions were conducted, with two groups consisting of six members and a third group consisting of seven members. An expert in the facilitation of focus groups was approached to act as a moderator for the sessions. Appointment of the moderator was important, as the researcher was a facilitator in the nursing college and known to the students, so this allowed participants to participate freely in the study. The moderator was a specialist psychiatric nurse skilled and experienced in focus group management. She is also an expert in qualitative research. The researcher made arrangements for the venue (a classroom in the Military Nursing College) in which to conduct interviews. The researcher also clarified the process of focus group interviews to the participants, as well as the expectations of the moderator during the interviews and how the interviews would be conducted. Data were recorded using a tape recorder with the permission of participants and field notes were handwritten by the researcher to capture actual discussions (Polit & Beck 2005:307). Participants' reactions and attitudes were also observed and noted throughout the interviews. Data were collected until saturation was reached (Streubert & Carpenter 2011:90–91).

## Data analysis

The data were analysed according to Henning's four-step method (2004:126). Tape-recorded interviews were listened to as soon as the interviews had been completed and these were then transcribed verbatim. The researcher recorded and organised the data on note cards and audiotapes were labelled to ease data retrieval. Cross-checking of data with each note card was performed to keep track of each piece of data collected. Units were then organised into a system derived from the data. After the data were organised they were ready to be categorised into themes and sub-themes. The researcher and co-coder created codes from the data independently; extracted codes were then compared for similarities and differences. Where differences occurred these were resolved, leading to emergence of the themes and sub-themes presented in the findings. The co-coder was a specialist in analysing and coding qualitative studies.

## Context of the study

The study was conducted in a Military Nursing College, of which there are three in South Africa. This college is situated in an urban area in one of the nine provinces in South Africa. A quiet classroom in one particular college was organised by the researcher in which to collect data without distraction.

# Ethical considerations

Approval to conduct this study was obtained from the Ethics Committee of the Faculty of Health Sciences of North-West University (Potchefstroom Campus), the Military Ethics Committee and the Officer Commanding of 3 Military Hospital. A written letter of request was given to the students, inviting them to participate in the study. Participants were informed in full about the study and were asked to provide written consent to participate. They were informed that the research did not form part of their work and that they could withdraw from participation at any time during the process without being disadvantaged in any way. All participants were assured that confidentiality would be maintained as only the researcher had access to the audiotaped material. Audiotapes were kept locked in a safe place and erased after the research was completed. The use of real names was avoided.

## Trustworthiness

Trustworthiness was maintained by using strategies of credibility, transferability, dependability and confirmability (Lincoln & Guba 1985:290). The research methodology and research context were described in detail to enable interested researchers to transfer these to other studies. The results and findings of the research process, including raw data, field notes, data reduction and analysis products, and theoretical notes relating to trustworthiness were kept as an audit trail of what transpired during the research process to ensure confirmability. Dependability was achieved through clear description of the exact methods of data collection, analysis and interpretation. The appointment of an independent coder with field research expertise also assisted in ensuring dependability. Prolonged engagement with the data assisted the researcher in identifying recurring patterns and themes, thus ensuring credibility.

# Results

This study set out to describe PEN experiences of facilitative and obstructive factors in clinical learning in two diverse settings. Two main themes emerged from the analysis: factors facilitating learning and those obstructing learning. Self-directed learning and acceptance by staff were experienced as facilitating PEN learning, whilst non-acceptance by staff, wearing of military uniform in public hospitals and workplace violence were experienced as obstructive to learning.

## Factors facilitating learning

### Self-directed learning

The limited number of learning opportunities in the military health setting afforded students ample time to practise procedures on these few available opportunities and to be in control of their own learning. The ability to control their own learning was perceived as empowering: they could make decisions and enjoy learning as they did not feel pressured to complete tasks but could work at their own pace. This practice made students feel that they are trusted to make judgements about patient care, which facilitated learning. This was represented in the following excerpts:

> 'Being a military nurse works at advantage because we are only serving military members and their family, so we don't have too much pressure on us, we learn at our own pace and given time to learn all of those stuff.' (PEN 1)
>
> 'They give us the opportunity to find solutions for patients' problems on our own and to make good judgements.' (PEN 8)
>
> 'Here at the military … we are given topics to prepare and present, we look for information ourselves; in that way we get to know what is happening in the ward.' (PEN 14)

### Acceptance by staff

Acceptance is another facilitating factor experienced by the students in this study. They expressed that they felt accepted by the doctors and nurses in the public CLE as they received good guidance and supervision from them in the wards of the public hospitals. This created confidence and competency in implementing nursing procedures, as the supervisor was always around. The following excerpts validate this:

> 'In the public hospital they accept us as part of the team, and the doctors will explain everything about the patient. We will do everything under supervision of the doctor.' (PEN 2)
>
> 'In the provincial hospital the sister calls you and teaches you the procedure.' (PEN 7)
>
> 'When we are in the outside hospitals and there are procedures that they want us to learn more about, they will call us to watch.' (PEN 10)

## Factors obstructive to learning

### Non-acceptance by staff

One respondent said that they did not always feel accepted, and was made to feel that they were always in the way, particularly in the military CLE:

> 'Sometimes they will become angry, saying students are just causing traffic. How could I learn when they don't accept us?' (PEN 11)

### Wearing military uniform in public health settings

Wearing military uniform when allocated to public hospitals was perceived as being obstructive to learning. PEN explained that they felt marginalised when referred to as soldiers and not nurses by some staff members. Their perception was that they were easily blamed for everything that went wrong because they wore a military uniform. The participants also alleged that some staff members often made condescending remarks about them and caused them a lot of embarrassment. Patient discomfort was also cited. Their views were stated as follows:

> 'Sometimes in public hospitals we feel bad as we wear military uniform, because they will call us soldiers and sometimes securities. They are sometimes not nice.' (PEN 9)
>
> 'Maybe something was not done, they will say "it's the soldiers"; it is always a pain for us, they call us soldiers and not nurses. So it's not nice as this embarrasses us.' (PEN 2)
>
> 'Patients are also not comfortable when we have to do procedures on them. They do not see us as nurses, they call us soldiers because of our uniform.' (PEN 15)

### Workplace violence

Participants expressed that they were often subjected to different forms of violence. These were mostly non-physical in nature, in the form of shouting, bad mouthing, being blamed and felt punished if one person was in the wrong. Some felt that military rules are applied in nursing and perceived this in a negative light:

> 'I just want to add on that issue of the shouting, wherever you go as long as you in the department they will shout.' (PEN 5)
>
> 'They call us, but when they call the college, stating how bad we are. The only thing we will hear from them, is how lazy we are, behind the staff nurses' back, they tell us we work more than them. They talk about us to the staff nurses, when those staff nurse are not there they talk about them.' (PEN 16)
>
> 'One of the old nurses forgot to take out the temperature, I mean, the thermometer, then when we were supposed to change the patient, eh, to give the patient a bed pan that thermometer fell and it broke, the glass was all over the bed. They blamed the students, even though we came after 3 \[*pm*\], they blame the students. If anything goes wrong they blame the students.' (PEN 13)
>
> 'He started swearing at me, verbally abusing me. I then called the police. I opened a case of assault.' (PEN 14)
>
> 'The military rules are applied in nursing and it is very destructive. When one student makes a mistake, everybody is punished.' (PEN 6)

One of the participants in this study was physically assaulted by a high-ranking member whilst carrying out his nursing duties:

> 'A rank is misused in the military; I was physically assaulted by a high-ranking member whilst carrying out my nursing duties.' (PEN 19)

# Discussion

Findings show that the students experienced the CLE as both facilitating and obstructive to their learning. Compared to the military setting, students experienced the public CLE to be more facilitative as they felt empowered, could self-direct their clinical learning and had support from the clinical staff. Silén and Uhlin (2008:462) are of the opinion that 'students' feelings of being in charge and having a genuine impact on the learning situations are crucial for their desire to take responsibility'. Compton (2005:36) asserts that there is a strong linkage between self-directed learning and positive psychology, and that attributes such as perceived control and satisfaction are important for self-directed learning to occur. Turner (2007) concurs that 'self-directed learners demonstrate greater awareness of their responsibility in making learning meaningful and monitoring themselves', thus making them more effective learners and social beings. According to Löfmark and Wikblad (2001:49), these positive attributes are said to be facilitating factors for learning in the clinical setting; these authors showed in their study that students value independence as a facilitating factor.

The second finding was that some participants experienced acceptance in the public setting where the staff was supportive and willing to supervise them. Several authors posit that it is important that students fit in and are accepted as part of the group during clinical placement, as this kind of welcoming environment is a source of active participation and provides learning opportunities (Hartigan-Rogers *et al*. 2007:1; Levett-Jones & Lathlean 2009:348; Nash, Lemcke & Sacre 2009:48). Staff members need to be available, friendly and willing to teach in order to construct a climate conducive to learning (Croxon & Maginnis 2009:240).

In their study, Levett-Jones and Lathlean (2008:107) found that 'students felt more empowered and enabled to capitalise on the available learning opportunities when they felt they had a legitimate place in the nursing team'; conversely, if not accepted, they can find it difficult to learn. Individuals' perceptions about their participation and level of acceptance in the group influence their sense of belonging (Tabane & Human-Vogel 2010:493). This sense of acceptance must be fostered, as learners who feel ostracised by those in authority may present with low self-esteem and unhappiness. If the PEN (learners) feel that they are not part of the collective, their functioning may not be optimal, resulting in a diminished learning process (Baumeister *et al*. 2002; Tabane & Human-Vogel 2010:504). According to Chesser-Smyth (2005:320), the morale of students can be boosted if they are welcomed, respected and valued and, as a result, they become more eager to learn.

It is also evident that students are not always accepted in the military CLE. Such behaviour is identified as being obstructive to learning (Hathorn, Machtmes & Tillman 2009:229).

Studies have shown that a lack of support in the CLE can be a source of anxiety and stress for students (Elliott 2002:72; Levett-Jones & Lathlean 2008:109; Papastavrou *et al*. 2009:176; Shipton 2002:244). Literature cites different reasons for a lack of support for students in the clinical setting, including registered nurses seeing facilitation of learning or supervision as extra work for them and the unavailability of staff (Clarke, Gibb & Ramprogus 2003:106; Mongwe 2001:4).

The wearing of military uniform was perceived as obstructive to learning by participants in the study. They claimed that the uniform caused them embarrassment, that they often felt marginalised and were blamed for multiple wrongs regarding patient care. Studies have shown that CLEs can be very traumatic for students when they are exposed to harsh remarks and made to feel like outsiders (Hathorn *et al*. 2009:242; Levett-Jones & Lathlean 2008:105; Rush, McCracken & Talley 2009:315).

The hierarchy within both CLEs made the PEN experience of clinical learning more challenging and was perceived as being obstructive to learning.

This study shows that participants were exposed to some form of workplace violence in the CLE. Workplace violence has many names in nursing literature. According to Mendez (2011:2), the phenomenon is known as horizontal violence, lateral violence, bullying, and intrapersonal workplace aggression. I the context of nursing, lateral violence has been defined as 'nurse-to-nurse aggression' (Griffin 2004:258). According to the literature, lateral violence finds expression in physical assault, psychological abuse and non-physical violence, such as verbal abuse, mobbing, gossip, jealousy, blaming, yelling, insulting, name-calling, lack of respect, bullying and intimidation, backstabbing, unfair criticism and defamatory statements about individuals or groups, whether it comes from nurses or other healthcare professionals (Bartholomew 2006:4–5; Brooks & Phillips 2013:9; Edwards & O'Connell 2007:27; Felblinger 2009; Gimeno *et al*. 2012: 30; Nachreiner *et al*. 2007:674)

Horizontal violence is defined as '\[*r*\]epeated, offensive, abusive, intimidating, or insulting behavior, abuse of power, or unfair sanctions that makes recipients upset and feel humiliated, vulnerable, or threatened, creating stress and undermining their self-confidence' (Vessey, DeMarco & Budin, as quoted in Sellers *et al*. 2009–2010:21). According to King-Jones (2011:80), horizontal violence includes forms of non-physical intergroup conflict that manifest in overt and covert behaviours of hostility ranging from intimidating body language to sarcastic comments, fault finding, belittling gestures, discourtesy, disinterest and discouragement, amongst other behaviours.

PEN were also subjected to physical violence. One of the participants was physically assaulted by a high-ranking member who was not satisfied with the treatment he received. According to Thomas and Burk (2009:229), this conduct can affect the working environment of the demoralised students and impede their learning. Experiences that have been described by PEN are related to the two very different roles – being a soldier and being a nurse – which causes the students to experience conflict. Fundamentally, one role is about force, aggression and violence whereas the other is about caring for the sick and the injured. It is not established if they have been prepared for these dual role.

# Limitations

There are three military nursing colleges in South Africa and the findings of the study are limited to the opinions of only one group of second-year students at one of these. It is therefore important to conduct future studies on students in other colleges.

# Conclusion

This study indicates that students experience both facilitating and obstructing factors to their learning during their clinical practice. The findings indicate that learning in the clinical setting is facilitated when students take responsibility and control of their learning and their self-confidence increases. Students feel safe to practise when they are supported by the clinical staff. They feel that they belong when the staff shows interest in them and welcomes them.

The study also showed that learning is obstructed when students are not accepted and are met with condescending comments or are blamed and embarrassed by the clinical staff. The lack of supervision in the military setting further obstructed their learning. Wearing of military uniform during the public clinical learning seemed to be problematic for the PEN. It was also evident that students experienced some forms of violence in the CLEs. Violence in any form is not conducive to learning and will be obstructive. Students cannot effectively be prepared clinically if the environment is not conducive to and supportive of clinical learning. Whilst research in this setting brought the experiences of military PEN to the fore, similar studies on non-military PEN may be useful in order to compare similarities and differences, with a view to using such information to improve the teaching and learning environment of students.

## Recommendations

Based on the conclusions of the study, it is recommended that students be allowed to take responsibility and initiative for their learning, as this can promote learning. It is also recommended that PEN should not be compelled to wear military uniform in public hospitals and should be allowed to dress in similar uniforms to their counterparts, so that they are not treated differently when allocated to the CLE of public hospitals. Clinical staff should be made aware of the impact that condescending remarks may have on students' learning. They should also recognise the need to support actively and welcome students so as to enhance the working environment. Policies on managing violence in the clinical area need to be developed, with specific attention on how this may affect students, particularly when they are dealing with high-ranked officers. There is also a need to prepare students for the dual role of being a nurse and a soldier so that they are able to experience the CLEs in a positive light.

## Acknowledgements

The authors would like to thank the Medical Research Council for funding the study.

## Competing interests

The authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article.

## Authors' contributions

E.M.C. (The South African Military Nursing College) conducted the study for her master's dissertation and provided input on the manuscript during the review process. E.L-M. (University of Limpopo) assumed the major responsibility for getting the article written and then corrected and refined the manuscript.

# References

[^1]: **How to cite this article:** Lekalakala-Mokgele, E. & Caka, E.M., 2015, 'Facilitative and obstructive factors in the clinical learning environment: Experiences of pupil enrolled nurses', *Curationis* 38(1), Art. \#1263, 7 pages. <http://dx.doi.org/10.4102/curationis.v38i1.1263>


abstract: In 1959 Ernst Mayr challenged the relevance of mathematical models to evolutionary studies and was answered by JBS Haldane in a witty and convincing essay. Fifty years on, I conclude that the importance of mathematics has in fact increased and will continue to do so.
author: James F Crow
date: 2009
institute: 1Genetics Laboratory, University of Wisconsin, 425 G Henry Mall, Madison, WI 53706-1574, USA
references:
title: Mayr, mathematics and the study of evolution

In 1959 Ernst Mayr (Figure 1) flung down the gauntlet \[1\] at the feet of the three great population geneticists RA Fisher, Sewall Wright and JBS Haldane (Figure 2): "But what, precisely," he said, "has been the contribution of this mathematical school to the evolutionary theory, if I may be permitted to ask such a provocative question?" His skepticism arose in part from the fact that the mathematical theory at the time had little to say about speciation, Mayr's major interest. But his criticism was more broadly addressed to the utility of the entire approach. A particular focus was the simplification that he called "beanbag genetics", in which "Evolutionary genetics was essentially presented as an input or output of genes, as the adding of certain beans to a beanbag and the withdrawing of others." \[1\].

Mayr was, however, criticizing textbook simplifications, rather than the actual work of the three pioneers. Far from treating gene frequency changes as analogous to the consequence of beans jostling at random in a bag, both Fisher and Wright considered gene interactions in detail. Fisher (Figure 2a) showed that, despite interactions between genes, natural selection acts on the additive component of the genetic variance. It is as if nature were familiar with least squares. The beanbag criticism was particularly inappropriate for Wright (Figure 2b), who specifically devised his 'shifting balance' theory as a way for a population to go from one harmonious gene combination (Mayr would say "integrated genotype") to another when intermediates were disadvantageous.

Who was to answer Mayr's criticism? Fisher was already dead, and in any case preferred attack to defense, and Wright was too gentle – though admittedly not always when Mayr was involved: returning from Italy where he had received the prestigious Balzan Prize in 1984, Wright told me that the value of the prize was considerably diminished when he discovered that Mayr had won it the year before. In the event, however, it was Haldane (Figure 2c) who took up the challenge. And he did it with flair and gusto. The result was "A defense of beanbag genetics" \[2\]. This was Haldane at his best – witty, spirited, informed, interesting and convincing.

But the larger question remains: what indeed has been the contribution of mathematical theory to evolution? Mathematics is not central to evolution in the way it has been in theoretical physics. Solid advances have been made without using mathematics, much being due to Mayr himself \[3\]. And these continue. Yet, I shall argue that mathematical ideas have made important, and often essential, contributions, and still do. Many concepts that are now established were arrived at mathematically, although their origins have since been forgotten.

For example, the idea that polymorphisms become stabilized in populations because heterozygotes are at an advantage is now found in elementary textbooks, but Fisher was the first to formulate it. Loss of heterozygosity with inbreeding is also textbook knowledge, but it was not clear until Wright developed the theory and invented a simple algorithm for quantifying it. Similarly, the idea that the impact of mutation on the population depends on the mutation rate rather than the magnitude of the mutant effect is now taken for granted, but that was not known until Haldane showed it mathematically. One final example is the inheritance of the ABO blood groups, which was in doubt from the time of their discovery at the turn of the twentieth century until Bernstein's mathematical population analysis in 1924 \[4\]. All of these applications used only elementary methods, and they must have been known to Mayr. Often, concepts that were developed mathematically were later explained in intuitive, non-mathematical ways. HJ Muller was particularly inventive in finding such explanations. But the mathematical derivation usually came first. It's a lot easier to find an intuitive explanation when you already know the answer.

Ironically, Mayr himself unwittingly provided an especially compelling argument for mathematical analysis. His theory of "genetic revolutions" assumed that from a well integrated population, genetic drift in a small founder offshoot will sometimes produce a population with a new set of genotypes integrated in a new way. Intuitively, a small founder population seemed a particularly unlikely place to find a new favorable gene combination, and this was indeed shown to be the case in a very detailed mathematical analysis by Barton and Charlesworth \[5\]. If Mayr had had more respect for mathematical population genetics, he never would have made what most theorists regard as the mistake of proposing that small founder populations are a likely source of major evolutionary changes by genetic drift.

Recent mathematical work has gone well beyond that of the three pioneers. Partly this is due to skilled mathematicians entering the field and bringing new techniques with them; especially noteworthy are stochastic processes. Second, and perhaps more important, is the extensive use of computers. Often you can use a computer to get by without deep mathematical knowledge. An additional influence is the explosive growth of molecular data, which lend themselves to mathematical treatment. In the first half of the twentieth century, population genetics and evolution had a beautiful theory, but there were very limited opportunities to apply it. Now the situation is reversed. Molecular data accumulate too fast to be assimilated.

What are some of the newer developments in evolution that are owed to mathematical theory? Here are a few.

# Neutral theory, molecular clocks and selective sweep

One striking result in the post-Mayr period was Motoo Kimura's neutral theory, independently developed in 1968 by him and by Jack King and Thomas Jukes \[6\]. These writers shocked the biological world by arguing that the bulk of molecular evolution is due to selectively neutral mutations driven by the mutation process rather than selection. I think it would please Mayr that the general idea – that the rate of evolution in the population is equal to the rate of mutation in a single individual – can be derived by simple reasoning using school mathematics. Yet, in order to apply the idea, we need to know how long a time period must be observed. This depends on how long it takes for a lucky new mutant to increase in frequency and completely replace its predecessors. That is not a simple problem and requires sophisticated theory. Kimura solved it using a diffusion model (see \[6\]). When selection and migration are taken into account, the theory is much more complicated.

One contribution of the neutral theory has been to provide a rationale for a molecular clock. Essentially, all our estimates of evolution rates depend on the assumption that the molecular changes used in constructing the clock are mutation-driven. The near constancy of average mutation rates permits reasonably accurate time estimates. Fortunately, enough of the DNA does not have an obvious function and can reasonably be supposed to be evolving by neutral kinetics, or near enough so that the neutral theory can be used in practice. And the experimenter can choose genomic regions most likely to behave in a neutral manner.

A second important attribute of the neutral theory is that it supplies a natural null hypothesis for the study of selection. And yet another outgrowth of the neutral theory is the view that much of the molecular polymorphism in natural populations is effectively neutral. This is especially useful now that variation in the frequencies of single-nucleotide polymorphisms (SNPs) is easily observed.

The various measures that are used to quantify genetic variability are outgrowths of population genetics theory. One striking result of such theory is the realization that all of the worldwide human population is descended from Africa, and moreover from a small area within Africa. The evidence for this striking conclusion is that molecular variance is greater in African peoples than elsewhere. The molecular clock can be used as one measure of the time taken during various human migrations and, of course, *Homo sapiens* is not the only species that can be studied in this way.

Another outgrowth of population thinking is the 'selective sweep'. A new favorable allele arises by mutation, spreads through the population and becomes fixed at a rate that is determined mainly by how favorable it is. A consequence of this fixation is that neutral or weakly selected alleles linked to the locus are swept along with it. Because of this, there is a region on either side of the selected locus that is deficient in genetic variability. Such regions of reduced variability are footprints of a selective sweep in the past and, remarkably, provide evidence for events that occurred long ago and which can no longer be observed. Although the basic idea is simple and requires no mathematics, an assessment of how much the variability is reduced and the linkage distance over which the reduced variability occurs depend on mathematical theory.

# Mathematics and the computation of family relationships

An area of biology in which mathematics, and especially computers, have become absolutely essential is systematics, Ernst Mayr's own field. Formerly, assessing species relationships and building phylogenetic trees based mainly on morphological differences was a matter of intuition and judgment. Systematists often disagreed, sometimes violently. Then came the DNA revolution. A mammalian DNA sequence supplies billions of bits of information, thus for the first time providing an opportunity for a procedure independent of personal judgments \[7\]. In recent decades the methods have steadily improved. The preferred procedures, such as Fisher's maximum likelihood, required a great deal of computation, and for a while this meant that large phylogenies were out of computer range. This is no longer true. Computers are now much faster, so their speed is no longer a limitation. Standards have increased in another way, too. It is now *de rigueur* to do statistical tests of significance of the tree structure and parts thereof. Many of these involve permutation methods, which have the merit of requiring minimum assumptions. They are computation-intensive, but with modern computers this is no longer an impediment.

One striking example from such studies, which came as a complete surprise to classical systematists, is the close relationship of the elephant to the shrew. Another example is in primates. For many decades the relationship of chimpanzee, gorilla and man has been uncertain. Molecular analysis of DNA sequences, using the newly developed theory, has shown that our closest relatives are chimpanzees. Furthermore, that we and the chimpanzees are 99% identical at the DNA level came as a surprise to many. Equally surprisingly, we share some 90% of our DNA with mice, rabbits, dogs, horses and elephants. Yet this is no surprise to those acquainted with the neutral theory. These numbers are fully consistent with expectations based on mutation rates and the times involved. Finally, there is now help available in the form of computer programs that can work out phylogenies and display the information graphically (see \[7\]). These not only eliminate a lot of tedious work, but place advanced methods in the hands of relative novices.

# Coalescence and speciation

Finally, there has been a major theoretical advance, coalescent theory \[8\]. Instead of looking forward in time, this method looks backward. Any two alleles or homologous nucleotides are ultimately derived from a single one; that is, looking backward, they coalesce. This has been the subject of extensive theoretical work in recent years. One problem for which coalescent theory provided at least an approximate answer is the question of whether there was any mating between our ancestors and contemporary Neanderthals. Small amounts of admixture are not ruled out, but coalescent theory has shown that any substantial intermating is very unlikely, as discussed by John Wakeley \[8\]. My other examples have been relatively simple, but this one isn't, as is apparent from this discussion. It involves a great deal of algebra. Another example, also given by Wakeley, is evidence for a selective sweep in *Drosophila simulans* \[8\].

Until recently, mathematical theory had contributed little to the study of speciation. Mayr emphasized allopatric speciation and the prevailing model, due to Dobzhansky and Muller \[9\], prevailed. Recent mathematical studies \[10\] support it and favor the view that speciation genes correspond to normal genes, selected for their effects within the species. Furthermore, there is evidence that these genes evolve rapidly. Thus, hybrid incompatibility is a by-product of ordinary selection in geographically isolated populations. There is no evidence that random drift plays an important part \[9\], so Mayr's 'genetic revolution' and similar ideas have little support. Yet it is important to point out that, aside from this, Mayr has usually been right \[3\]. The field of mathematical studies of speciation is barely started; it will surely increase.

# Looking to the future

I have given only a few examples of the part that mathematical theory has played in evolution studies. There are many more, but these, I hope, constitute a convincing sample of the importance of mathematics in population genetics and evolution. I do not intend to imply that all evolutionary study need be mathematical and theory-driven. Much exciting evolutionary biology is done in the Mayr non-mathematical tradition \[3\]. For example, 'evo-devo' studies, looking at changes in development during evolution, have produced exciting results while largely ignoring population genetics. Another non-mathematical example is horizontal gene transfer brought about by transposable elements, which is especially important in the evolution of microorganisms. There is also abundant evidence for increases in genetic complexity by the accumulation of small duplications. And, as always, a lot of morphological and behavioral evolution is interesting in and of itself. Yet, my guess is that as these subjects become more quantitative, population genetic theory will play an increasing role.

The rise of molecular methods has led to an increase in the importance of mathematics in population genetics and evolution. The abundance of data that require mathematical analysis has greatly increased. At the time of Mayr's challenge, evolution had a beautiful theory but very few opportunities to apply it. Now the situation is reversed: data appear faster than existing theory can deal with them. That mathematics will play an increasingly important evolutionary role in the near future seems clear.

# Envoi

I think these examples show not only that mathematical theory is helpful, but that it is often essential. I don't know what Ernst would say today. He might have had a change of mind, but I doubt it. Knowing how much he enjoyed arguing, I suspect he would be quite critical of much that I have written. Unfortunately, although he lived to be 100, he was not immortal and died in 2005. Were he still alive, I would surely hear from him and whatever his opinions, he would not keep them to himself. He would have enjoyed an argument, preferably over a glass of sherry. And so would I.

### Acknowledgements

I am indebted to Bret Payseur for reading the manuscript and offering some very useful suggestions.


abstract: # Introduction
  .
  Neutrophils and monocytes play an important role in overt inflammation in chronic inflammatory joint diseases such as rheumatoid arthritis (RA). The sympathetic nervous system (SNS) inhibits many neutrophil/monocyte functions and macrophage tumor necrosis factor (TNF), but because of the loss of sympathetic nerve fibers in inflamed tissue, sympathetic control is attenuated. In this study, we focused on noradrenergic and TNF regulation of human neutrophil peptides 1-3 (HNP1-3), which are proinflammatory bactericidal α-defensins.
  .
  # Methods
  .
  Synovial tissue and cells were obtained from patients with RA and osteoarthritis (OA). By using immunohistochemistry and immunofluorescence, HNP1-3 were tracked in the tissue. With synovial cell-culture experiments and ELISA, effects of norepinephrine, TNF, and cortisol on HNP1-3 were detected.
  .
  # Results
  .
  HNP1-3 were abundantly expressed in the synovial lining and adjacent sublining area but not in deeper layers of synovial tissue. The human β-defensin-2, used as control, was hardly detectable in the tissue and in supernatants. HNP1-3 double-stained with neutrophils but not with macrophages, fibroblasts, T/B lymphocytes, and mast cells. Norepinephrine dose-dependently decreased HNP1-3 levels from RA and OA cells. TNF also inhibited HNP1-3 levels from OA but not from RA cells. Cortisol inhibited HNP1-3 levels only in OA patients. A combination of norepinephrine and cortisol did not show additive or synergistic effects.
  .
  # Conclusions
  .
  This study demonstrated an inhibitory effect of norepinephrine on HNP1-3 of mixed synovial cells. In light of these findings, the loss of sympathetic nerve fibers with low resting norepinephrine levels might also augment the inflammatory process through HNP1-3.
author: Birgit Riepl; Susanne Grässel; Reiner Wiest; Martin Fleck; Rainer H Straub
date: 2010
institute: 1Laboratory of Experimental Rheumatology and Neuroendocrino-Immunology, Division of Rheumatology, Department of Internal Medicine I, University Hospital, F.J. Strauss Allee 11, 93053 Regensburg, Germany; 2Department of Orthopedic Surgery, University Hospital Regensburg, Asklepios Clinic Bad Abbach, Kaiser-Karl-V.-Allee 3, 93077 Bad Abbach, Germany
references:
title: Tumor necrosis factor and norepinephrine lower the levels of human neutrophil peptides 1-3 secretion by mixed synovial tissue cultures in osteoarthritis and rheumatoid arthritis

# Introduction

Rheumatoid arthritis (RA) is a chronic joint disease leading to severe erosions of adjacent bone, which are not observed in patients with osteoarthritis (OA). Although an inflammatory process is present in OA synovial tissue, RA patients demonstrate a higher state of synovial tissue inflammation compared with OA patients. In the pathophysiology of RA, T cells, B cells, macrophages, fibroblasts, and osteoclasts play dominant roles. In addition, neutrophils are important mediators of tissue inflammation in RA, and neutrophils are the most abundant cell type in the synovial fluid \[1\]. Neutrophil production of proteases, reactive oxygen species, S100 proteins, cytokines, chemokines, and complement stimulate inflammation \[2\]. Fc-gamma receptors on neutrophils can bind immune complexes that can perpetuate the inflammatory process \[2\]. In addition, neutrophils produce important antimicrobial proteins, such as defensins \[3\].

A large number of defensins and defensin-like peptides have been reported in many organisms. As of March 2010, 363 entries had been recorded in a defensin database \[4\]. One distinguishes α-defensins (in neutrophils) from β-defensins (in other cells). Human neutrophils contain four α-defensins (HNP-1 to HNP-4) \[5\]. HNPs are unique to neutrophils and account for \~99% of the total α defensin content of these cells \[5\]. HNPs exert chemotactic, immunomodulating, and cytotoxic effects and participate in inflammation \[5\]. In contrast, human β-defensins have been described mainly in epithelial cells but also in leukocytes, heart, skeletal muscle, testis, keratinocytes, tonsil, placenta, and other tissues \[4\]. Defensins are expressed and released on bacterial stimuli involving the Toll-like receptors and, in addition, other stimuli, such as cytokines \[6\]. Besides microbicidal activities, defensins can stimulate TNF secretion from macrophages, as recently reported \[7\].

In tissues of patients with RA and OA, the investigation of defensins has recently begun. One report demonstrated the presence of HNP1 3 positive cells in synovial tissue of healthy subjects and in patients with suppurative arthritis, osteoarthritis (OA), and RA \[8\]. However, regulation of HNP1-3 in these diseases (for example, by cytokines) has not yet been investigated. Another report found an association of HNP1-3 in synovial fluid of patients with RA and severe erosive joint disease \[9\]. These studies clearly demonstrate that neutrophil defensins are present in inflamed tissues of patients with RA and OA. Some reports also demonstrated that defensins can be produced by dendritic cells and monocytes \[10,11\], but this has not been demonstrated in synovial tissue or cells of RA and OA patients.

For several years, we have been interested in the role of the sympathetic nervous system (SNS) in OA, RA, and experimental arthritis \[12,13\]. We have been attracted by effects of the SNS on neutrophils and monocytes, but effects on defensin secretion are presently not known. It is recognized that activation of the SNS enhances neutrophil and monocyte mobilization, leading to increased numbers of circulating neutrophils and monocytes, called the first line of defense \[14-16\]. Such a mechanism might increase the numbers of neutrophils and monocytes that enter inflamed tissue at sites of leaky endothelial structures (leakiness is important because catecholamines inhibit neutrophil and monocyte attachment to normal endothelium). However, once neutrophils and monocytes have entered inflamed tissue, the major neurotransmitter of sympathetic nerve fibers, norepinephrine, inhibits several neutrophil and monocyte functions. For example, norepinephrine decreases migration \[17,18\], oxygen radical production \[19\], phagocytosis \[18\], and bactericidal activity \[20\]. These inhibitory influences were present only when norepinephrine appeared at high concentrations (via β~2~-adrenergic receptors). This might be quite different at low concentrations when norepinephrine exerts its effects via α-adrenoceptors \[21-23\].

Thus, in the presence of sympathetic nerve fibers in the tissue, norepinephrine would inhibit many proinflammatory activities of neutrophils and monocytes via β~2~-adrenoceptors, which might also play a role in synovial tissue of patients with OA and RA. However, in inflamed synovial tissue of patients with RA, sympathetic nerve fibers are lost and replaced by catecholamine-producing cells \[24\]. The remaining catecholamine concentrations are low in the synovial tissue, leading to concentrations suitable only for α-adrenergic signaling \[24\]. Although norepinephrine through α-adrenergic signaling stimulates proinflammatory factors in neutrophils and macrophages \[21-23\], these low concentrations may well play a proinflammatory role in inflamed tissue.

By using synovial cells of patients with RA, we investigated the effect of norepinephrine on abundance of the proinflammatory bactericidal proteins HNP1-3. Experiments were also carried out in cells from OA patients because possible differences might represent one factor for the differential effects on bone (erosions in RA versus formation of new bone in OA). For comparison, we investigated the human defensin β-defensin 2 (HBD 2). TNF was used as another important proinflammatory stimulus to influence defensin secretion for comparison. In addition, the study aimed to investigate the influence of cortisol alone or together with norepinephrine because cooperative antiinflammatory effects of cortisol and norepinephrine have been described \[25\].

# Materials and methods

## Patients

In this investigation, we included 10 patients with OA and seven patients with RA. Diagnosis of RA was based on the established criteria according to the American College of Rheumatology (formerly, the American Rheumatism Association) \[26\]. The characteristics of patients are given in Table 1. Erythrocyte sedimentation rate and serum levels of C-reactive protein were measured by using standard techniques.

Patient characteristics

|  | Osteoarthritis | Rheumatoid arthritis |
|----|----|----|
| Number | 10 | 7 |
| Age (years) | 62.8 ± 3.2 | 57.0 ± 4.0 |
| Sex (f/m) | 6/4 | 4/3 |
| C-reactive protein (mg/L) | 5.1 ± 1.3 | 25.9 ± 14.1^\#^ |
| Erythrocyte sedimentation rate (mm) | 11.5 ± 1.5 | 27.6 ± 3.1\* |
| Lining layer thickness, cells | 2.0 ± 0.5 | 2.5 ± 0.2 |
| Cellular density (all cells), cells/mm^2^ | 364 ± 169 | 635 ± 329 |
| T-cell density, cells/mm^2^ | 1.1 ± 0.1 | 1.9 ± 0.5 |
| Macrophage density, cells/mm^2^ | 55 ± 38 | 52 ± 16 |
| Medication |  |  |
|  Prednisolone (%) | N.A. | 86.0 |
|  Daily prednisolone (mg/day) | N.A. | 6.1 ± 1.7 |
|  Methotrexate (%) | N.A. | 71.0 |
|  Leflunomide (%) | N.A. | 14.0 |
|  Nonsteroidal antiinflammatory drugs (%) | 70.0 | 71.0 |

Data are given as mean ± SEM. \**P* \< 0.05; ^\#^*P* \< 0.005 for the comparison with osteoarthritis. Abbreviations: N.A., not applicable.

The study was approved by the Ethics Committee of the University of Regensburg. Patients were informed about the purpose of the study and gave written consent.

## Synovial tissue preparation, isolation, and culture of primary mixed synovial cells

OA and RA patients underwent elective knee-joint replacement surgery. Synovial tissue samples were obtained immediately after opening the knee-joint capsule, preparation of which was described \[24\]. In brief, a piece of synovial tissue of ≤9 cm^2^ was dissected. A larger piece of the synovial tissue was used to isolate mixed synovial cells (for culture experiments, see later). Approximately eight pieces of the same synovial area were used for immunohistochemistry and immunofluorescence, which were fixed for 12 to 24 hours in phosphate-buffered saline (PBS) containing 4% formaldehyde and then incubated in PBS with 20% sucrose for 12 to 24 hours. Thereafter, they were placed in protective freezing medium and quick-frozen (Tissue Tek; Sakura Finetek, Zoeterwoude, The Netherlands). All tissue samples were stored at 80°C.

For culture experiments, mixed synovial cells were isolated during the morning hours by enzymatic digestion for 1 to 2 hours at 37°C by using Liberase (Roche Applied Science, Mannheim, Germany). At approximately 2 to 4 p.m., the synovial cells were resuspended in RPMI 1640 medium (Sigma, Taufkirchen, Germany), supplemented with 1% penicillin/streptomycin (Life Technologies, Inc., Paisley, U.K.) and 0.1% amphotericin B (Bristol-Myers Squibb, Munich, Germany). In total, 2.5 × 10^5^ isolated synovial cells of OA or RA patients were incubated for 24 hours in the presence of 200 μM L-ascorbic acid, together with norepinephrine, TNF, cortisol, or norepinephrine plus cortisol in indicated concentrations (all substances from Sigma, Steinheim, Germany).

The percentage of different types of synovial cells was tested by specific antibodies against prolyl 4 hydroxylase (for the synoviocyte type B, fibroblasts; Calbiochem, Bad Soden, Germany), CD163 (synoviocyte type A, macrophages; Dako, Hamburg, Germany), CD3 (T cells; Dako), CD19 (B lymphocytes; Dako) neutrophils (elastase; Fitzgerald Industries Int. Inc., Acton, MA, USA), and mast cells (tryptase; Abcam, Cambridge, UK). In preliminary experiments with primary early-culture mixed synoviocytes, we detected that \~37% were positive for prolyl 4 hydroxylase, 26% for CD163, 12% for CD3, 5% for CD19, 10% for elastase, and \<1% for tryptase.

## Immunohistochemistry and double immunofluorescence

Approximately ten 5-μm sections were cut from the frozen tissue blocks. For immunohistochemistry with alkaline phosphatase as the enzyme system, sections were blocked with 20% acetic acid for 20 minutes at 4°C. Sections were further blocked with 10% bovine serum albumin, 10% fetal calf serum, and 10% chicken serum for 45 minutes (all from Sigma). Then, sections were incubated with either monoclonal mouse anti-human antibodies against HNP1-3 (BMA Biomedicals, Augst, Switzerland, no. T 1034; dilution, 1:1,000; these antibodies recognize HNP-1 to -3) or polyclonal rabbit anti-human antibodies against HBD-2 (Biologo, Kronshagen, Germany, no. DEF002; dilution, 1:100). Both primary antibodies were incubated overnight at 4°C. After intensive washing with phosphate-buffered saline, sections were incubated with secondary antibodies for 1 hour at room temperature (for HNP1-3, goat anti-mouse coupled to alkaline phosphatase; Dako, no. D0486; dilution, 1:100; for HBD-2, goat anti-rabbit coupled to alkaline phosphatase, Dako, no. D0487; dilution, 1:100). The sections were developed by using BCIP/NBT substrate (Dako, K0598). We controlled specific staining by using irrelevant primary antibodies or serum or by omitting the primary antibody.

Double immunofluorescence was carried out with fixed frozen tissue and a similar blocking procedure to that mentioned earlier. For HNP1-3 and HBD-2, the primary antibodies of BMA Biomedicals and Biologo were used in the same dilution as given earlier. For double staining, respective antibodies were used against activated macrophages (CD163, Dako), T lymphocytes (CD3, Dako), fibroblasts (prolyl-4 hydroxylase, Dako), B lymphocytes (CD19, Dako), mast cells (tryptase; Abcam, Cambridge, UK), and neutrophils (elastase; Fitzgerald Industries International; and Lab Vision NeoMarkers via Thermo Scientific, Dreieich, Germany). Fluorescent staining of positive cells was achieved by incubating the sections with respective secondary Alexa Fluor 488 and 546 antibodies or F(ab')~2~ fragments (Molecular Probes via Invitrogen, Karlsruhe, Germany). Nuclei were stained with Vectashield mounting medium with DAPI (Vector Laboratories via Biozol, Eching, Germany). We controlled specific staining by using irrelevant primary antibodies or serum or by omitting the primary antibody.

## Determination of HNP1-3 and HBD-2 in supernatants of synovial cells

HNP1-3 were measured with commercially available ELISA (HyCult Biotechnology, Uden, The Netherlands; this ELISA recognizes HNP-1 to -3). The detection limit of this assay was 20 pg/ml. Intra- and interassay coefficient of variation were \<10%.

With respect to HBD-2, a new ELISA was established by using two commercially available antibodies (capture: Biologo, no. DEF002, polyclonal rabbit anti-human antibodies; dilution, 1:1,000; detection, R&D Systems, Wiesbaden, Germany; AF2758, polyclonal goat anti-human antibodies; dilution, 1:200). After overnight coating with the capture antibody at 4°C, extensive washing, and blocking with 10% fetal calf serum, 100 μl of standard (recombinant HBD-2; Dianova, Hamburg, Germany, no. CYT-26732) or 100 μl supernatant of synovial cells was incubated for 2 hours at room temperature. After extensive washing, the detection antibody was added for another hour (at room temperature). After extensive washing, a rabbit anti-goat tertiary antibody was used, coupled to biotin (Dako, Hamburg, Germany; no. E0466). By using streptavidin coupled to horseradish peroxidase and tetramethylbenzidine (TMB) as the substrate, the ELISA was developed. The detection limit of this assay was \~8 pg/ml. Intra- and interassay coefficients of variation were \<15%.

## Superfusion technique of synovial tissue

As described in detail earlier \[24\], we used a microsuperfusion-chamber apparatus to superfuse pieces of synovial tissue with culture medium. This technique allows the determination of spontaneous defensin release directly from fresh synovial tissue. The superfusion chambers had a volume of \~80 μl. Super-fusion was performed for 6 hours at a tempera-ture of 37°C and a flow rate of 66 μl/min. Synovial tissue pieces had a standard size of 5 μm in diameter with a precision biopsy punch (Stiefel, Offenbach, Germany). Every hour, superfusate was collected to measure HNP1-3 and HBD-2, as described earlier.

## Presentation of the data and statistical analysis

All data are given as mean ± SEM. Box plots give the 10^th^, 75^th^, 50^th^ (median), 25^th^, and 10^th^ percentile. Group medians were compared by using the nonparametric Mann-Whitney test (SPSS/PC, Advanced Statistics, V15.0, SPSS Inc., Chicago, IL, USA). A value of *P* \< 0.05 was the significance level.

# Results

## Immunohistochemical localization of HNP1-3 and HBD-2 and double immunofluorescence

To study the localization of defensins, immunohistochemistry of the synovial lining and sublining area was performed in patients with OA and RA. A representative staining of an RA patient is given in Figure 1. HNP1 3 were detected mainly in the synovial lining and in the directly adjacent sublining area (Figure 1, left panels). HNP1-3 were present in most of the RA patients and OA patients. This was quite different for HBD-2, which was rarely detectable in RA and OA patients. The staining in Figure 1, right panels, is a rare example of an RA patient in whom HBD-2-positive cells were detected in the sublining zone.

In further extensive double immunofluorescence studies, we provide evidence that HNP1-3 were colocalized to elastase-positive neutrophils (Figure 2, left panels). HNP1-3 were not detected in macrophages, fibroblasts, T lymphocytes, B lymphocytes, or mast cells. Similar to that in immunohistochemistry, HBD-2 was rarely detected with immunofluorescence. In these cases, HBD-2 was colocalized with prolyl-4 of fibroblasts and CD19 of B lymphocytes (Figure 2, right panels). No similar colocalization was observed for macrophages, T lymphocytes, neutrophils, or mast cells.

## HNP1-3 and HBD-2 from synovial tissue and synovial cells

To study the release of HNP1-3 or HBD-2, the protein was detected in the superfusate of synovial tissue. Synovial tissue released HNP1-3, which was particularly evident in RA patients (Figure 3A). The slow decline of superfusate concentration is typical for a washout of the protein and not for a decrease in secretion or production (Figure 3a). Compatible with the findings in immunohistochemistry, HBD-2 superfusate levels were lower, as substantiated in RA and OA patients (Figure 3b).

On a very different time scale, levels of defensins were studied in supernatants of cultured mixed synovial cells (Figure 3c). The levels of HNP1-3 increased over time, whereas HBD-2 was barely detectable (Figure 3c, compare detection limits with those in Figure 3a, b). Because HNP1-3 were detectable by ELISA, further functional studies included only these peptides. We studied early HNP1-3 appearance within 24 hours to minimize a possible effect by necrosis or apoptosis of these cells.

## Density of neutrophils in synovial tissues of patients with OA and RA

The presence of HNP1-3 prompted us to study the density of elastase-positive neutrophils in synovial tissues of patients with OA and RA. The density of neutrophils was higher in RA as compared with OA patients (Figure 3d). The relatively low density of neutrophils is due to the fact that neutrophils were present mainly in the lining and adjacent sublining area but not in deeper layers of the synovial tissue, which were also considered in calculating tissue density (see legend in Figure 3d).

## Influence of TNF, norepinephrine, and cortisol on levels of HNP1-3

To study important factors that might influence HNP1-3 levels, such as proinflammatory cytokines like TNF, this cytokine was used in culture experiments with mixed synoviocytes. TNF slightly decreased HNP1-3 levels in the supernatant of OA cells, which was significant at 1 ng/ml (Figure 4). In RA synoviocytes, TNF did not exert a similar U-formed dose-response effect because no significant differences were observed (Figure 4).

In contrast, norepinephrine decreased supernatant HNP1-3 levels produced by mixed synovial tissue cultures in both OA and RA patients (Figure 5a, b). It is obvious that this inhibition was present only at high concentrations when norepinephrine exerts its effects mainly via β-adrenoceptors (Figure 5a, b).

In OA patients, cortisol at high concentrations of 10^-6^ *M* reduced HNP1-3 concentrations (Figure 5c), which was not observed in RA patients (Figure 5d). In addition, the combination of cortisol plus norepinephrine led to a decrease of HNP1-3 levels produced by mixed synovial tissue cultures in OA and also in RA patients (Figure 5c, d). However, the combined effect of the two hormones did not exceed the individual effects of norepinephrine alone.

# Discussion

This study demonstrates the presence of two human defensins in the synovial tissue of patients with OA and RA. The α-defensin HNP1-3 and the β-defensin HBD-2 were present in synovial tissue, whereas HNP1-3 was undoubtedly allocated to neutrophils (we have not tested for dendritic cells). HBD-2 was found in a small number of fibroblasts and B lymphocytes. Although HNP1-3 were easily detectable by using histologic techniques or in culture experiments, HBD 2 was barely visible in tissue and measurable in supernatants. This study further demonstrated that norepinephrine inhibited HNP1 3 in both RA and OA patients' mixed synovial cell cultures, and cortisol and TNF slightly inhibited this α-defensin only in OA patients. This investigation adds to the understanding of how the SNS might influence HNP1-3 in chronic inflammatory joint diseases.

In a previous report, the presence of HNP1 3-positive cells was demonstrated in the synovial lining of healthy subjects and in patients with suppurative arthritis, OA, and RA \[8\]. However, these authors did not study the regulation of HNP1-3, which was a particular aspect in the present work. Another study reported very high synovial fluid levels of HNP1 3 in RA patients, associated with more-severe erosions \[9\]. Because we used the same ELISA mentioned in the latter study, we were surprised that our superfusate and supernatant levels of HNP1-3 were lower. We interpret these earlier findings of Bokareva *et al.* \[9\] insofar as HNP1-3 accumulate in the synovial fluid, which has been demonstrated for other factors as well (for example, estrogens \[27\]). Because we have not studied synovial fluid levels, we cannot directly compare the results of the two studies. However, discrepant findings might also depend on increased abundance of neutrophils in synovial fluid, as compared with synovial tissue.

Although HNP1-3 defensins have been reported to be expressed by granulocytes in synovial tissue \[8\], it was not clear whether these proteins are actually released in the tissue. By using the superfusion approach, we were able to demonstrate that these proteins are released by few granulocytes in synovial tissue. In the present study, HNP1-3 is produced in higher amounts in RA than in OA, which might reflect the overall greater inflammation in patients with chronic RA. This is particularly relevant because RA patients were treated with prednisolone and antiproliferative drugs that should have reduced proinflammatory factors like HNP1-3. HNP1-3-positive cells were elastase-positive neutrophils, and we can exclude that other cells such as macrophages, fibroblasts, T and B lymphocytes, or mast cells stain positive for this protein. However, we have not tested dendritic cells that can also produce HNP1-3 \[10,11\]. The relatively low number of neutrophils in the tissue might be a consequence of rapid migration into the synovial fluid (\>95% are neutrophils).

The mentioned first study of Paulsen *et al.* also reported on HBD-2 \[8\], which was not found in their samples investigated (they found the defensins HBD-3, CAP37, and LL37). We confirmed that immunohistologic detection of HBD-2 is a rare event (found in only one patient with RA and in one with OA). Nevertheless, this RA patient had numerous positive cells that allowed double staining, revealing double-positive fibroblasts and B lympho-cytes. Because HBD-2 levels were below the detection limit in most superfusate and supernatant samples, functional experiments with TNF and hormones aimed only to investigate the α-defensin HNP1-3. Further studies corroborated that neutrophils are present in OA and RA tissue, and that the density of these cells is higher in RA than in OA patients. It is known that the SNS has a strong influence on neutrophils (but also on monocytes), so we were particularly interested in the functional effects of the SNS on HNP1-3.

The major neurotransmitter of sympathetic nerve fibers, norepinephrine, inhibits several cellular functions. For example, norepinephrine decreases migration \[17,18\], oxygen radical production \[19\], phagocytosis \[18\], and bactericidal activity \[20\]. These inhibitory influences on neutrophils (but also on monocytes) were present only when norepinephrine appeared at high concentrations (via β~2~-adrenergic receptors). This might be quite different at low concentrations, when norepinephrine exerts its effects via α-adrenoceptors \[21-23\]. Our present study supports the inhibitory influence of norepinephrine, focusing on the α-defensin HNP1-3. In both RA and OA patients, norepinephrine dose-dependently decreased supernatant levels of HNP1-3 from mixed synovial tissue cultures, which reached the significance level only at high concentrations. These norepinephrine effects were stronger when compared with cortisol alone, which did not influence HNP1-3 in RA synovial cells. In addition, the combination of norepinephrine plus cortisol did not materialize in an additive or even synergistic effect. These results confirm the overall inhibitory effect of norepinephrine on cellular functions, given that concentrations of this neurotransmitter are high enough. At low concentrations of 10^-9^ *M* norepinephrine, we did not observe opposite effects that would indicate a proinflammatory influence on mixed synovial tissue cultures via α-adrenoceptors.

Because TNF is an important proinflammatory molecule in chronic inflammatory joint diseases, the effect of this cytokine on HNP1-3 levels was tested in this study. It was known that β-defensins such as HBD-2 can be stimulated by TNF in epithelial cells and astrocytes \[28,29\], but, to our knowledge, effects of TNF on HNP1-3 were not investigated (especially not in OA/RA material). TNF decreased HNP1-3 supernatant levels in OA mixed synovial cultures, and the dose-response curve was U-shaped with a maximum inhibition at 1.0 ng TNF/ml. TNF did not exert similar effects in RA cell cultures, which may be related to already high TNF levels in these cell preparations (receptor desensitization). Because, conversely, HNP1-3 can stimulate TNF secretion from macrophages, as recently reported \[7\], the TNF-induced inhibition of HNP1-3 might be part of a negative-feedback loop that prevents overshooting of innate immune responses. From this point of view, we hypothesize that HNP1-3 precedes TNF in the stimulatory cascade of innate immunity events. This would be an important aspect because TNF has been installed at the forefront of the innate immunity cascade in RA \[30\].

# Conclusions

In conclusion, HNP1-3 is present in neutrophils, and as compared with HBD-2, it is abundantly present in chronically inflamed synovial tissue of patients with RA and OA. Because HNP1-3 levels are decreased by norepinephrine in mixed synovial-cell cultures, this study demonstrates that the SNS might inhibit neutrophil (but also monocyte) function *in vivo*. Sympathetic nerve fibers are lost in inflamed tissue \[24,31\], so concentrations of this neurotransmitter are probably too low to induce antiinflammatory activities. A similar situation exists for TNF, which is also decreased by norepinephrine \[32\]. HNP1-3 can stimulate secretion of TNF \[7\], so the loss of sympathetic nerve fibers with low concentrations of norepinephrine might have double-negative effects, possibly increasing both local HNP1-3 and TNF levels. This scenario can be relevant in overt inflammation of the tissue when monocytes (HNP1-3, TNF), macrophages (TNF), and neutrophils (HNP1-3) play a decisive role.

# Competing interests

The authors declare that they have no competing interests.

# Authors' contributions

BR and SG participated in the concept and design and acquisition of data. RW and MF participated in interpretation of data and in drafting and revising the article. RHS participated in the concept and design, analysis and interpretation of data, and drafting and revising the article.

## Acknowledgements

The authors thank Dr. Sven Anders and Dr. Joachim Grifka for kindly supplying synovial tissue.

This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Research Unit FOR696).