End-point effector stress mediators in neuroimmune interactions: their role in immune system...

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Please note: Images will appear in color online but will be printed in black and white.ArticleTitle End-point effector stress mediators in neuroimmune interactions: their role in immune system homeostasis

and autoimmune pathologyArticle Sub-Title

Article CopyRight Springer Science+Business Media, LLC(This will be the copyright line in the final PDF)

Journal Name Immunologic Research

Corresponding Author Family Name DimitrijevicParticle

Given Name MirjanaSuffix

Division Immunology Research Center “Branislav Jankovic”

Organization Institute of Virology, Vaccines and Sera, “Torlak”

Address 458 Vojvode Stepe, 11221, Belgrade, Serbia

Email [email protected]

Author Family Name StanojevicParticle

Given Name StanislavaSuffix




Email

Author Family Name KustrimovicParticle

Given Name NatasaSuffix




Email

Author Family Name LeposavicParticle

Given Name GordanaSuffix

Division Department of Physiology, Faculty of Pharmacy

Organization University of Belgrade

Address Belgrade, Serbia

Email

ScheduleReceived

Revised

Accepted

Abstract Much evidence has identified a direct anatomical and functional link between the brain and the immunesystem, with glucocorticoids (GCs), catecholamines (CAs), and neuropeptide Y (NPY) as its end-point. Thissuggests the important role of these mediators in immune system homeostasis and the pathogenesis ofinflammatory autoimmune diseases. However, although it is clear that these mediators can modulatelymphocyte maturation and the activity of distinct immune cell types, their putative role in the pathogenesisof autoimmune disease is not yet completely understood. We have contributed to this field by discoveringthe influence of CAs and GCs on fine-tuning thymocyte negative selection and, in particular, by pointing tothe putative CA-mediated mechanisms underlying this influence. Furthermore, we have shown that CAs areimplicated in the regulation of regulatory T-cell development in the thymus. Moreover, investigations relatedto macrophage biology emphasize the complex interaction between GCs, CAs and NPY in the modulationof macrophage functions and their putative significance for the pathogenesis of autoimmune inflammatorydiseases.

Keywords (separated by '-') Glucocorticoids - Catecholamines - Neuropeptide Y - Thymocyte negative selection - T regulatory cells -Macrophages - Autoimmune diseases - Experimental autoimmune encephalomyelitis

Footnote Information Mirjana Dimitrijevic, Stanislava Stanojevic, and Gordana Leposavic equally contributed to this work.

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IMMUNOLOGY IN SERBIA1

2 End-point effector stress mediators

3 in neuroimmune interactions: their role in immune

4 system homeostasis and autoimmune pathology

5 Mirjana Dimitrijevic • Stanislava Stanojevic •

6 Natasa Kustrimovic • Gordana Leposavic

7

8 � Springer Science+Business Media, LLC 2012

9 Abstract Much evidence has identified a direct anatomical and functional link between the brain and the immune system,

10 with glucocorticoids (GCs), catecholamines (CAs), and neuropeptide Y (NPY) as its end-point. This suggests the important

11 role of these mediators in immune system homeostasis and the pathogenesis of inflammatory autoimmune diseases.

12 However, although it is clear that these mediators can modulate lymphocyte maturation and the activity of distinct immune

13 cell types, their putative role in the pathogenesis of autoimmune disease is not yet completely understood. We have

14 contributed to this field by discovering the influence of CAs and GCs on fine-tuning thymocyte negative selection and, in

15 particular, by pointing to the putative CA-mediated mechanisms underlying this influence. Furthermore, we have shown

16 that CAs are implicated in the regulation of regulatory T-cell development in the thymus. Moreover, investigations related

17 to macrophage biology emphasize the complex interaction between GCs, CAs and NPY in the modulation of macrophage

18 functions and their putative significance for the pathogenesis of autoimmune inflammatory diseases.

19

20 Keywords Glucocorticoids � Catecholamines � Neuropeptide Y � Thymocyte negative selection � T regulatory cells �

21 Macrophages � Autoimmune diseases � Experimental autoimmune encephalomyelitis

22

23 Introduction

24 For many years, the central nervous system (CNS) and the

25 immune system were considered entirely autonomous body

26 systems. However, during the last 50 years, a wealth of

27 clinical and experimental evidence demonstrating bidirec-

28 tional communications between these two body systems

29 has been accumulated. Consequently, a rapidly expanding

30area of research called neuroimmunomodulation, which

31explores the cellular and molecular bases of neuroimmune

32communications, and the significance of this crosstalk for

33immune system homeostasis and pathologies involving

34immune cell-mediated mechanisms, has been established.

35Twenty-five years ago, Professor Branislav D. Jankovic

36wrote a historical mini-review devoted to the 30th anni-

37versary of the research activities of the so-called ‘‘Belgrade

38Neuroimmunomodulation Group’’ at the Immunology

39Research Center in Belgrade [1]. This review provided one

40of the first integrative views of comprehensive neuroim-

41mune interactions. An explosive increase in the number of

42scientists committed to researching neuroimmune interac-

43tions led to the point in 2007 when Hugo Besedovsky and

44Andriana del Rey, on the 20th anniversary of the Brain

45Behavior and Immunity journal, posed the question of

46whether the time had arrived to re-name this field by

47simply calling it ‘‘Integrative Biology and Medicine’’ [2].

48However, in spite of tremendous advancements in under-

49standing neuroimmune interactions and their significance

A1 Mirjana Dimitrijevic, Stanislava Stanojevic, and Gordana Leposavic

A2 equally contributed to this work.

A3 M. Dimitrijevic (&) � S. Stanojevic � N. Kustrimovic

A4 Immunology Research Center ‘‘Branislav Jankovic’’, Institute

A5 of Virology, Vaccines and Sera, ‘‘Torlak’’, 458 Vojvode Stepe,

A6 11221 Belgrade, Serbia

A7 e-mail: [email protected];

A8 [email protected]

A9 G. Leposavic

A10 Department of Physiology, Faculty of Pharmacy,

A11 University of Belgrade, Belgrade, Serbia

Mirjana Dimitrijevic

123Journal : Large 12026 Dispatch : 21-2-2012 Pages : 17

Article No. : 8275h LE h TYPESET

MS Code : IMRE123 h CP h DISK4 4

Immunol Res

DOI 10.1007/s12026-012-8275-9

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50 for immune system homeostasis, alostasis and dystasis,

51 many important issues still need to be targeted.

52 It has been generally accepted that the main stress

53 effector axis, i.e., hypothalamic–pituitary–adrenal (HPA)

54 axis, and sympathetic limb of the autonomic nervous sys-

55 tem provide the most important communication avenues in

56 neuroimmune interactions [3]. Thus, many research

57 endeavors have been devoted to defining their role in

58 immune system homeostasis and immunopathology. The

59 Belgrade Neuroimmunology Group spearheaded by Pro-

60 fessor B.D. Jankovic played a pioneering role in this

61 research.

62 This manuscript briefly outlines recent findings related

63 to the role of mediators, forming the major interface

64 between the brain and immune system in establishing

65 thymic central tolerance and macrophage functions. It also

66 points to the putative significance of alterations in the

67 neuron-immune interface for the development of inflam-

68 matory autoimmune diseases.

69 Stress and autoimmunity

70 The stress system, which activates when the complex

71 dynamic body equilibrium (homeostasis) is challenged by

72 internal or external adverse forces, encompasses a central

73 and peripheral stress effector system [3]. The central part

74 of this system includes the hypothalamic, corticotropin-

75 releasing hormone (CRH) and the locus ceruleus and

76 autonomic NE centers in the brainstem. The peripheral part

77 of the stress system comprises glucocorticoids (GCs),

78 whose secretion is regulated by the hypothalamic–pitui-

79 tary–adrenal (HPA) axis, and the catecholamines (CAs),

80 i.e., NE and epinephrine, which are released from the

81 peripheral sympathetic nerve endings and adrenomedullary

82 cells. Neuropeptide Y (NPY), which is co-released with NE

83 from the sympathetic nerve endings and adrenomedullary

84 cells upon stress-induced activation of the sympatho-

85 adrenal system [4], also constitutes a component of this

86 system [5]. The two main stress effector axes are shown to

87 interact at multiple levels, modulating each other’s effects

88 on target cells, including the immune system cells. Their

89 end-point mediators, such as GCs, CAs and NPY, have

90 long been considered to exert immunosuppressive effects

91 on the immune system. However, mounting experimental

92 and clinical evidence currently supports their immuno-

93 modulatory action [6–9]. In this line are findings showing

94 the pro-inflammatory and anti-inflammatory actions of

95 NPY, depending on the interaction between different Y

96 receptor subtypes in relation to the cell type, age, and

97 animal strain [10–17].

98 Given that stress has been related to the development of

99 many inflammatory autoimmune diseases [18], the role of

100the end-point effector stress mediators in the pathogenesis

101of these diseases has been extensively investigated [19–

10221]. The role of the stress mediators in the development of

103multiple sclerosis (MS) has been particularly well docu-

104mented [22]. MS is an inflammatory and degenerative

105disease of CNS, with an assumed autoimmune-mediated

106pathogenesis [23, 24]. Stress has been recognized as one of

107the trigger factors in the onset and/or relapse of MS

108symptoms [25–29]. However, a protective role for stress in

109MS has also been suggested [30, 31]. Many of the clinical,

110histological, immunopathogenic and pharmacological

111characteristics of human MS can be observed in its’ animal

112model, for example, experimental autoimmune encepha-

113lomyelitis (EAE). Depending on the immunization proto-

114col, specificity of the neuroantigen and the animal strain,

115various forms of EAE differing in clinical appearance of

116the disease (acute, chronic, and relapsing form) can be

117established (reviewed in [32]). The central role in the

118pathogenesis of EAE is ascribed to MHC class-II-restricted

119CD4 ? T lymphocytes [33]. Immunization with a spinal

120cord homogenate or myelin basic protein in adjuvant

121activates peripheral myelin-specific CD4 ? T cells that

122have escaped immune tolerance. The priming and prolif-

123eration of myelin-reactive CD4 ? T cells in the peripheral

124lymphoid organs precedes their migration to the CNS.

125Infiltrating CD4 ? T cells are reactivated through inter-

126action with local antigen-presenting cells in the CNS [34].

127They secrete chemokines that primarily recruit macro-

128phages to the inflammation site. Macrophages in turn

129secrete pro-inflammatory cytokines such as TNF-a and IL-

1301, which are important for both perpetuating inflammation

131and contributing to CNS tissue damage [35].

132As mentioned, data concerning the effect of stress on the

133onset and severity of EAE are inconsistent [36–44]. The

134influence of stress on EAE depends on several factors,

135including the specificity of stressors, genetic traits of the

136animal strain, duration of stress, and temporal relationship

137between the exposure to stress and induction of EAE.

138Studies from other research groups have demonstrated that

139stress experienced in early life such as maternal depriva-

140tion, premature weaning, and neonatal exposure to noise,

141handling or gentling primarily increased susceptibility to

142EAE in adult animals [39, 41, 42, 44–46]. Although the

143molecular mechanisms behind this phenomenon are elu-

144sive, the findings that exposure to various stressors in the

145critical neonatal period modify susceptibility to EAE later

146in life [47, 48] are related to long-lasting disturbances in

147HPA activity, including both hyper- and hypo-responsive-

148ness [49]. In the same vein are data showing that rats

149exhibiting similar susceptibility to the induction of EAE,

150e.g., Lewis and Dark Agouti (DA) rats, display differential

151changes in HPA activity in response to various stressors

152[50–52]. Thus, it may be speculated that any deviations

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153 from HPA normoactivity could contribute to the disease

154 development.

155 Apart from the role in the mounting response to self-

156 antigens in EAE, GCs have been shown to play a role in the

157 regulation of the autoimmune response. This is corrobo-

158 rated by the findings indicating that: (1) corticosterone

159 plasma levels increase and decrease during the peak and

160 resolution of the disease, respectively [53]; (2) corticoste-

161 rone replacement not only reduces the rate of EAE, but also

162 the severity of the disease in adrenalectomized rats before

163 induction of the disease [54]; and (3) a drop in the plasma

164 corticosterone level below physiological levels after the

165 initial attack is associated with relapses of EAE [55]. The

166 reduced HPA axis responsiveness has been found in MS

167 patients with secondary progressive and relapsing/remitting

168 disease [31]. However, elevated basal plasma levels of

169 cortisol and ACTH, indicating hyperactivity of the HPA

170 axis, in MS patients have also been reported [56]. There-

171 fore, in MS, as in EAE, changes in HPA activity are sug-

172 gested to be associated with the development of disease.

173 Moreover, in MS, apart from changes in HPA activity,

174 alterations in CA action have been observed. Specifically,

175 an increased number of b-adrenergic receptors (ARs) on

176 lymphocytes [57, 58] and impaired intracellular signal

177 transduction [59] have been reported. The density of b-ARs

178 is also increased on lymphocytes from rats and mice

179 developing EAE [60, 61], whereas no data exist showing

180 their deregulated intracellular signaling. In addition, the

181 altered production of CAs in the peripheral blood mono-

182 nuclear cells of patients suffering from MS suggests the

183 involvement of immune cell-derived CAs, acting in an

184 autocrine/paracrine manner, in the development of relapses

185 and remissions of the disease [62–64].

186 Elucidation of the role of GCs and CAs in EAE and MS

187 is additionally complicated, since GCs are implicated in the

188 regulation of both CA synthesis [65] and b-AR expression

189 on lymphocytes [66]. It has been hypothesized that the

190 worsening of acute EAE and increased mortality in adre-

191 nalectomized animals [67, 68] is a consequence not only of

192 reduced peripheral GC levels but also of related alterations

193 in CA action. Our research group showed that chronic

194 blockade of b-ARs with propranolol starting 4 days before

195 immunization prevented adrenalectomy-induced aggrava-

196 tion of the disease course without affecting mortality [69].

197 In addition, the b-AR blockade suppressed the develop-

198 ment of clinical symptoms and reduced the number of

199 lesions in the spinal cord and cerebellum of EAE rats. This

200 finding contradicts the previously reported detrimental

201 impact of chemical sympathectomy on EAE [70, 71].

202 Several lines of evidence can explain the observed differ-

203 ences between the effects of these two procedures used for

204 the disruption of CA action. First, chemical sympathec-

205 tomy induces the depletion of CAs and other sympathetic

206mediators such as NPY and ATP, which may also be

207involved in the modulation of EAE. Moreover, the sup-

208pressive action of exogenous NPY has been demonstrated

209in EAE mice [72]. Specifically, NPY decreased the severity

210of clinical signs of EAE, presumably by decreasing IFN-c

211secretion from autoreactive T lymphocytes and elevating

212the IgG1-IgG2a ratio of autoantigen-specific antibodies,

213which is indicative of favoring the Th2 response. The

214agonists of Y1 receptor also inhibited the induction of

215EAE, thereby emphasizing the role of Y1 receptors in the

216suppressive effect of NPY on EAE [72]. Furthermore,

217blocking the Y1 receptor signaling immediately after

218immunization for EAE led to an earlier onset of the dis-

219ease. The latter finding indicates that endogenous NPY

220plays a protective role in EAE induction [72]. In the same

221vein are data showing reduced concentrations of NPY in

222plasma and cerebrospinal fluid of patients with MS [73].

223However, their relevance for the onset and course of MS is

224unknown. Therefore, the aggravation of EAE following

225chemical sympathectomy is unlikely caused by decreased

226levels of NPY and is mostly related to CA insufficiency

227[70, 71]. Second, blockade of b-AR does not interrupt the

228effect of CAs mediated via a-adrenergic receptors. More-

229over, lymphocytes and monocytes normally express only

230b-ARs, whereas the expression of a-AR is induced during

231inflammatory autoimmune diseases [74–76]. Therefore, an

232opposing role for a- and b-ARs in the CA-induced modu-

233lation of EAE is suggested. Several findings also point to

234the mechanism underlining the effects of chronic b-AR

235blockade on the development of EAE. Given that b-ARs

236are reported to participate in the CAs’ effects on leukocyte

237mobilization and trafficking [7, 77, 78], their chronic

238blockade could hamper the migration of lymphocytes and

239macrophages into the CNS. Additionally, our research has

240shown that b-AR blockade reduces the percentage of

241peripheral CD4 ? CD25 ? T lymphocytes [69]. Since the

242CD4 ? CD25 ? cell population includes both activated

243and regulatory T-cells (Tregs), two putative explanations

244for protective action of chronic b-AR blockade on EAE can

245be suggested. First, considering that NO is shown to sup-

246press the expansion of antigen-specific T lymphocytes

247during immune response activation [79] and was found to

248increase NO production in macrophage following chronic

249b-AR blockade [80], it may be assumed that the b-AR

250blockade, which increases the NO production in these cells,

251decreases the percentage of activated CD4 ? CD25 ? T

252lymphocytes. Second, it may be hypothesized that b-AR

253blockade affects the number of activated T-cells and Tregs

254in an opposing manner. Specifically, it reduces the overall

255number of activated cells and increases number of sup-

256pressive Tregs. The latter assumption is supported by

257recent reports showing that peripheral sympathectomy

258protects mice from EAE by increasing the number of Tregs





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259 in their spleen and lymph nodes due to an induction of

260 Tregs from naive T-cells [81].

261 The altered HPA axis and SNS activity seem to be

262 related to the onset and regulation of the autoimmune

263 response; many significant pieces in the complex mosaic of

264 events governing autoimmune responses, leading to tissue

265 lesions and the consequent development of inflammation,

266 are still missing. Although the different types of immune

267 cells and molecular mechanisms are involved in the path-

268 ogenesis of autoimmune diseases, including MS/EAE, our

269 research has mainly been focused on the influence of the

270 end-point stress mediators on the thymic mechanisms

271 providing central tolerance and modulation of the periph-

272 eral macrophage secretory and antigen-presenting

273 functions.

274 Alterations in the brain–immune system interface

275 and autoimmunity

276 End-point stress effector mediators and thymic central

277 tolerance

278 The hallmark of T-cell central tolerance is clonal deletion,

279 viz. T-cell receptor (TCR)-mediated apoptosis of poten-

280 tially hazardous T-cell progenitors with a high affinity for

281 self-antigens [82]. The mechanisms that eliminate (clonal

282 deletion and lesser important receptor editing) or func-

283 tionally impair (cell anergy) high-affinity self-reactive cells

284 are assigned as negative selection mechanisms (‘‘reces-

285 sive’’ tolerance) [reviewed in 83]. In addition, to secure the

286 self-tolerance, the thymus provides a positive selection of

287 CD4 ? CD25 ? FoxP3 ? T regulatory cells (Tregs) [84],

288 natural killer T (NKT) cells [85], and CD8aa ? intestinal

289 intraepithelial lymphocytes [86]. These cells in the

290 periphery actively restrain self-reactive T-cells that have

291 escaped thymic negative selection (‘‘dominant’’ tolerance)

292 (reviewed in [83]).

293 The affinity of the TCR for self-peptide–MHC ligands

294 expressed by thymic stromal cells is the crucial parameter

295 that drives the developmental outcome in the thymus

296 (reviewed in [83]). Thymocytes that have no or very low

297 affinity die by neglect. If the TCR has a low affinity for

298 self-peptide–MHC, then the thymocytes survive and dif-

299 ferentiate (positive selection). If the progenitor has a high

300 affinity for self-peptide–MHC, then at least two outcomes

301 are possible [83]. First, the cell can be subjected to negative

302 selection. Second, it is likely that there are mechanisms to

303 select high-affinity self-reactive cells and subsequently

304 provide differentiation into cells that express the ‘‘regula-

305 tory’’ phenotype. It is still unclear what determines whether

306 a T-cell is tolerized by negative selection or is selected to

307 become a regulatory T-cell.

308Even though it is well established that the mechanisms

309of ‘‘recessive’’ (that is, cell intrinsic) and ‘‘dominant’’ (that

310is, trans-acting) central tolerance greatly reduce the threat

311of autoimmunity inherent in the adaptive immune system,

312the exact role of the thymus in mounting and regulating

313autoimmune responses in the context of composite inter-

314play between thymus and the peripheral immune system is

315still poorly understood. Several lines of evidence support

316the role of the thymus in the pathogenesis of autoimmune

317diseases. It has been shown that: (1) thymectomy in adult

318patients with myasthenia gravis is associated with

319increased risk of the development of systemic autoimmune

320conditions [87], while thymectomy on postnatal day 3 leads

321to the induction of multiple organ-specific autoimmune

322diseases [88] and (2) the altered composition of the

323peripheral Treg subset related to a reduced thymic-depen-

324dent de novo generation of recent thymic emigrants (RTE)-

325Treg with a compensatory expansion of memory Treg may

326contribute to the Treg suppressive deficiency associated

327with MS [89]. Furthermore, data indicate that: (1) EAE

328recovery is preceded by thymic changes mirrored in

329accelerated differentiation and the proliferation of Tregs

330through a mechanism involving the increased expression of

331IL-7 in stromal cells and dynamic expression of the IL-7

332receptor in thymic Treg cells [90] and (2) thymectomy in

333young rats and mice prevents spontaneous recovery from

334EAE [90, 91].

335Stress and thymus

336The thymus is shown to respond to various insults,

337including environmental stressors, infection and a wide

338variety of substances, by transient atrophy [92, 93]. The

339histological changes substantiating this accidental atrophy

340are similar to those characterizing age-associated thymic

341atrophy (thymic involution). Accidental thymic atrophy has

342been related to an increased risk of autoimmunity [94].

343However, in light of data indicating that diminished thy-

344mopoiesis due to thymic atrophy leads to the homeostatic

345proliferation of T-cells in periphery that, in turn, may

346increase the opportunity for self-reactive T-cells to expand

347and consequently induce autoimmune disorders [95], a

348question may arise concerning the contribution of impaired

349efficiency of central tolerance mechanisms to stress-induce

350autoimmune pathology. To understand the role of the

351thymus in stress-induced changes in susceptibility and the

352development of autoimmune diseases, it is necessary to

353identify putative relevant thymic changes and then to prove

354their significance for autoimmune pathology. Concerning

355stress-induced thymic atrophy, it should be taken into

356account that the increased secretion of CAs and GCs is the

357major trademark of the prototypical stress response [96].

358Therefore, discovering the cellular and molecular events





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359 underlying thymopoietic changes in stress-induced thymic

360 atrophy requires one to formulate the influence of these

361 mediators on T-cell development. This is corroborated by

362 the data showing that thymic cells express the receptors for

363 CAs and GCs. The receptors for GCs were detected in

364 thymic epithelial cells [97, 98] and thymocyte subsets at

365 distinct stages of differentiation and maturation [99–101].

366 In addition, GCs are shown to exert effects on the thymus

367 not only through the nuclear translocation of the ligand-

368 bound GR from the cytoplasm, which then acts as a tran-

369 scription factor, but also through a putative membrane GR

370 (reviewed in [102]). Moreover, direct membrane and

371 mitochondrial effects of GCs have been suggested

372 (reviewed in [102]). Our research has contributed to map-

373 ping the thymic cells expressing ARs by demonstrating the

374 expression of a1b-ARs on rat thymic cells [103] and by

375 identifying lymphoid and non-lymphoid thymic cell sub-

376 sets bearing a1b- and b2-ARs [103–106].

377 The role of GCs in thymic atrophy supports data

378 showing that: (1) adrenal insufficiency in humans and

379 adrenalectomy in experimental animals lead to thymic

380 hypertrophy [107–112] and (2) adrenalectomy-induced

381 thymic hypertrophy cannot be reversed by epinephrine

382 (normally produced by the adrenal medulla) [107–109].

383 Data also show that: (1) adrenalectomy prevents stress and

384 drug-induced involution of the thymus [110] and (2)

385 administration of adrenocorticotropic hormone (ACTH) to

386 mice caused a marked reduction in thymic weight [113].

387 Furthermore, our research showed that adrenalectomy

388 affected T-cell development in DA rats and identified the

389 thymocyte developmental stages, which were sensitive to

390 the lack of circulating GCs [112]. In addition, significant

391 evidence shows that GR- and TCR-signaling ‘‘crosstalk’’

392 determines thymocyte destiny in terms of survival [114–

393 116].

394 Concerning CA’s influence on the thymus, an early

395 study of Sylvie Durant showed that administration of the b-

396 AR agonist, isoproterenol, led to a significant decrease in

397 thymic weight and an even greater decrease in thymocyte

398 numbers and that hydrocortisone augmented these effects

399 of isoproterenol [117]. Our research demonstrated that

400 long-term treatment with urapidil, an a1-AR antagonist,

401 increased thymic weight and cellularity in young rats [103].

402 More importantly, in vitro [118, 119] and whole-animal

403 studies [103, 120, 121] revealed the significant role of CAs

404 in T-cell differentiation/maturation. In addition, they have

405 suggested that age-related changes in CA signaling may be

406 implicated in the development of thymic involution, which

407 is in similar to accidental thymic atrophy [122, 123]. It has

408 been demonstrated that CAs, acting via b-and a1-ARs,

409 influence the TCR-dependent stages of T-cell development,

410 including those involving the selection process [120, 122,

411 123]. In addition, in our hands, the long-lasting blockade of

412b- and/or a1-ARs affects the thymic production of regula-

413tory cells [120, 122]. Furthermore, our research showed

414that adrenalectomy augments the effects of b-AR blockade

415with propranolol on T-cell differentiation/maturation [124].

416Moreover, it suggested that adrenalectomy could influence

417the efficiency of thymic a1-AR-mediated signaling [105,

418122]. Considering the aforementioned, one may speculate

419that the end-products of the main effector components,

420which often act in concert in stress [96], may affect the

421thymic mechanisms of immunological tolerance.

422Glucocorticoids

423An accumulating body of evidence [114–116, 125–128],

424including those provided by our study [112], points to the

425role of GCs in thymocyte selection. This is supported by

426data showing a crosstalk between GR- and TCR-signaling

427termed ‘‘mutual antagonism.’’ The first evidence in favor of

428this concept, according to which the outcome of the

429selection processes depends on GC/TCR interplay, came

430from studies indicating that the GR- and TCR-signaling

431independently induce apoptosis in T-hybridoma cells, yet

432together promote the survival of these cells [125, 126]. It

433has been assumed that GCs play a decisive role in ‘‘death

434by neglect’’ of thymocytes expressing TCRs with no or

435very low affinity for self-peptide–MHC. In addition, they

436interfere with TCR-induced death signaling in cells with

437TCRs of intermediate affinity, enabling these cells to

438escape TCR-induced apoptosis and undergo positive

439selection. In addition, it has been suggested that the TCR

440activation-induced apoptotic signaling in thymocytes

441bearing TCRs with a high affinity for self-peptide–MHC

442overwhelms GC antagonism, resulting in the deletion of

443these cells, i.e., negative selection [114]. Therefore, a

444logical implication of the ‘‘mutual antagonism’’ concept is

445that high levels of GCs affect the survival of self-reactive

446T-cells, which, in turn, may predispose for the development

447of autoimmunity. However, it should be pointed out that

448although pharmacological and adrenal gland ablation

449studies support the aforementioned relationship between

450GR- and TCR- signaling, data obtained in genetically

451modified mouse models investigating the GR function in

452T-cells have generated opposing findings [112, 129, 130].

453A fully convincing explanation for these conflicting data

454has not yet been offered. The findings indicating non-

455receptor-mediated and non-genomic effects of GCs at

456higher hormone concentrations [131–133] may provide a

457plausible explanation. It has been also speculated that GR-

458signaling is dispensable for thymocyte development and

459homeostasis under steady-state conditions [116].

460If one accepts the previous scenario of GC action on

461developing T-cell survival, then molecular pathways of the

462GC/TCR interplay remain to be articulated. Although the





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463 putative molecular mechanisms involved in the crosstalk

464 between GR- and TCR-signaling are still largely unex-

465 plored, some indicative findings exist. It was shown that:

466 (1) a proapoptotic BH3-only protein Bim is required for the

467 initiation of apoptosis induced by GCs and (2) activation of

468 the extracellular signal-regulated kinase (ERK) pathway

469 promotes phosphorylation and the proteasome-dependent

470 degradation of Bim [134, 135]. These findings, aggregately

471 with those indicating that the development of T-cell

472 resistance to dexamethason requires a strong TCR-signal-

473 ing and activation of the ERK signaling pathway [136], can

474 be associated with GC inefficiency in antagonizing strong

475 TCR-signaling in thymocytes. On the other hand, it was

476 shown that GC acting at the membrane level may cause the

477 rapid dissociation of the lymphocyte-specific kinases lck

478 and fyn from the TCR complex, thus inactivating TCR

479 signaling [137]. Alternatively, it has been suggested that

480 CGs, acting via genomic and mitochondrial pathways, may

481 influence the balance between pro-apoptotic and anti-

482 apoptotic members of the Bcl-2 family, i.e., so-called Bcl-2

483 survival rheostat (reviewed in [102]), and consequently

484 prime the thymocytes for subsequent selection signaling

485 [116]. This is corroborated by data indicating that distinct

486 thymocyte subsets express GRs at different levels and

487 exhibit differential sensitivity to GC action [99].

488 Moreover, it should be highlighted that the outcome of

489 GC/TCR interplay is believed to depend on local thymic

490 GC synthesis [114–116]. The synthesis of this steroid has

491 been proven in thymic epithelial cells [138–140] and thy-

492 mocytes entering the selection processes, as well as in

493 CD4 ? CD8 ? post-selected CD69 ? thymocytes [141,

494 142]. GC production in thymic epithelial cells exhibits an

495 age-associated decrease, whereas DP thymocytes increas-

496 ingly secrete GC with age [116, 142]. This implies dif-

497 ferential regulation of GC synthesis in different thymic cell

498 types. Furthermore, the ectopic thymic GC synthesis

499 imposes a question on the relationship between adrenal-

500 and thymus-derived GCs. It seems particularly justifiable in

501 light of our findings indicating that adrenalectomy affects

502 not only overall thymic cellularity, but also the efficacy of

503 thymocyte selection [112]. Contrary to expectations, it was

504 shown that the removal of adrenal GCs by adrenalectomy

505 elevated plasma ACTH levels, which, in turn, inhibited GC

506 production by thymocytes [116]. This is related to thymic

507 hypercellularity in adrenalectomized animals and suggests

508 a role of thymocyte GCs in the regulation of thymocyte

509 ‘‘death by neglect’’ and, possibly, positive selection [116].

510 Conversely, ACTH was shown to enhance GC synthesis in

511 thymic epithelial cells [143]. This finding leads to a new

512 question on the relationship between the GCs from distinct

513 thymic cellular sources.

514 Finally, our research pointed out that the withdrawal of

515 circulating CGs enhances the effects of b-AR blockade on

516thymocyte differentiation and negative selection [122] by

517diminishing thymic NE levels and altering thymocyte

518sensitivity to CA action [144]. Accordingly, we hypothe-

519sized that thymic NE may act as a putative mediator in

520modulation of the thymocyte selection by adrenal GCs.

521Concerning the impact of GCs on the thymic production

522of regulatory T-cells, findings show that GCs do not

523influence the generation of thymic NKT cells [145].

524However, no data exist on the influence of GCs on the

525maturation of Tregs in the thymus.

526Collectively, the data summarized in this section and

527schematically outlined in Fig. 1 indicate that alterations in

528adrenal and thymic GC action may affect immunological

529tolerance at the thymic level by modulating the efficacy of

530the negative selection. However, although much data have

531been collected, the composite picture of GC molecular

532interactions influencing this complex process has not yet

533been deconvoluted.

534Catecholamines

535Our investigations have demonstrated that long-term

536treatment with propranolol, a b-AR antagonist, in rats of

537different strains such as Wistar, Albino Oxford (AO) and

538DA rats, leads to substantial changes in the distribution of

539the main thymocyte subsets delineated by simultaneous

540analysis of CD4, CD8, and TCRab expression [120, 146,

541147]. The altered relationship between thymocyte subsets

542suggests impaired negative thymocyte selection. The same

543model system demonstrated that b-AR blockade increases

544Thy-surface density on thymocytes exposed to selection

545pressure [120]. Thy-1 (CD90), originally named theta (h)

546antigen, then Thy-1 (THYmocyte differentiation antigen

5471), due to its prior identification in thymocytes, is a small

548glycosyl phosphatidylinositol-anchored protein found in

549rodents on T-cells, thymocytes, and neurons [148]. It is

550particularly abundant on the thymocyte surface [149]. Thy-

5511 expression increases with thymocyte maturation, reach-

552ing the maximum at the CD4 ? CD8 ? double positive

553stage of T-cell development, and decreases with further

554maturation [149]. This molecule has been conserved

555throughout evolution, suggesting its important function

556[150]. Two lines of evidence directly relate altered Thy-1

557expression on thymocytes to impaired negative selection in

558propranolol-treated rats. Firstly, it was shown that thymo-

559cytes from Thy-1-/- mice had altered cell–cell contacts,

560and hyper-responsiveness to TCR triggering, as demon-

561strated by the heightened activation of p56lck, phosphory-

562lation of TCR subunits, Ca2? fluxes and cell proliferation

563[151]. In addition, analysis of thymopoiesis in these ani-

564mals demonstrated impaired maturation of thymocytes

565from the double positive to single positive stage of devel-

566opment, due to inappropriate negative selection. In other





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567 words, these observations indicate that Thy-1 negatively

568 regulates TCR-mediated signaling and thereby controls

569 thymocyte selection [151]. Secondly, it has been shown

570 that: (1) exogenous cAMP and NE decrease steady-state

571 Thy-1 mRNA levels in murine thymocytes or S49 mouse

572 thymoma cells through post-transcriptional destabilization

573 of Thy-1 mRNA and (2) the NE-mediated decreases in

574 Thy-1 mRNA levels are prevented by propranolol [152].

575 Recent investigations identified an AUUUA regulatory

576 element (ARE), a motif commonly associated with mRNA

577decay, in Thy-1 mRNA. They also showed that ARE acts

578as a destabilizing element in the presence of cAMP [153].

579More specifically, it was demonstrated that cAMP signal-

580ing led to Thy-1 mRNA decay, through protein kinase

581A-mediated phosphorylation of ARE binding protein HuR,

582and a consequent decrease in the ability to stabilize this

583mRNA. Thus, it seems obvious that changes in b-AR-

584mediated NE signaling may affect thymocyte negative

585selection by altering the strength of TCR-signaling via

586modulation of the Thy-1 surface density (Fig. 1). In

Fig. 1 Schematic outline of putative cellular and molecular mech-

anisms implicated in glucocorticoid (GC) and catecholamine (CA)-

mediated modulation of thymic negative selection and production of

CD4 ? CD25 ? FoxP3 ? T regulatory (Tregs) and natural killer T

(NKT) cells. Circulating GCs, by altering thymic GC production in

thymic epithelial cells and thymocytes, as well as CA (norepineph-

rine, NE) release from sympathetic nerve fibers and CA-synthesizing

thymic stromal cells and thymocytes/or expression of adrenergic

receptors (AR) on these cells may affect the efficiency of thymocyte

negative selection (1). The ultimate outcome of direct GC action on

thymocyte negative selection depends on the complex interaction

between GC signaling via the intracellular and membrane glucocor-

ticoid receptor (GR) and TCR signaling. It was shown that GCs,

acting at the membrane level, may cause the rapid dissociation of the

lymphocyte-specific kinases lck and fyn from the TCR complex,

leading to a disruption of TCR signaling. In addition, membrane and

intracellular GR-mediated signaling, through genomic and mitochon-

drial pathways, may influence the balance between pro-apoptotic and

anti-apoptotic members of the Bcl-2 family (Bcl-2 rheostat), and

thereby prime the thymocytes for the subsequent selection signal. On

the other hand, TCR signaling may impair the GC influence on the

Bcl-2 rheostat, via activation of an extracellular signal-regulated

kinase pathway that promotes the phosphorylation and proteasome-

dependent degradation of proapoptotic protein Bim. Acting via b-

ARs, CAs are shown to affect negative selection (1) via down-

regulation of Thy-1 (CD90) expression on CD4 ? CD8 ? double

positive (DP) thymocytes, and consequently the potentiation of TCR

signaling. In addition, CAs, via the b-AR–mediated mechanisms,

diminish the thymic production of CD4 ? CD25 ? FoxP3 ? T

regulatory cells (Tregs) (2), while engaging in both b- and a1-AR-

mediated mechanisms, they reduce NKT cell generation (2)





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587 addition, the authors’ recent study investigating the influ-

588 ence of chronic treatment with urapidil, an a1-AR antag-

589 onist, on T-cell development, suggests that this treatment

590 might also diminish thymocyte negative selection, but most

591 likely, via a different mechanism [122]. Collectively, the

592 previous findings suggest that an insufficient thymocyte

593 negative selection due to a stress-induced increased NE

594 level is not likely to occur.

595 However, our research group demonstrated that chronic

596 b-AR blockade with propranolol increases thymic pro-

597 duction of Tregs [120], while long-term treatment with

598 urapidil augments the production of NKT cells [122]

599 (Fig. 1). Thus, it may be assumed that NE exerts a tonic

600 inhibitory influence on the thymic production of these cells.

601 Although Tregs also arise in the periphery [154], the

602 increased thymic generation of Tregs, following b-AR

603 blockade, is in accordance with data showing a robust

604 increase in the number of CD4 ? FoxP3 ? Tregs in the

605 spleen, following the ablation of sympathetic innervations

606 in the periphery by the neurotoxin 6-hydroxydopamine

607 [81]. The increased proportion and number of CD4 ? cells

608 expressing a regulatory phenotype in the thymi from rats

609 subjected to b-AR blockade may be linked to data showing

610 that: (1) mature Tregs constitutively express tyrosine

611 hydroxylase (TH), the rate-limiting enzyme in the synthesis

612 of CAs, and contain substantial amounts of dopamine, NE,

613 and epinephrine, which could act in an autocrine/paracrine

614 manner [155]; (2) the most mature thymocytes were shown

615 to express TH [156], and (3) CAs induce thymocyte

616 apoptosis acting in an autocrine manner [157]. In other

617 words, increased Treg neogenesis following propranolol

618 treatment may reflect the decreased thymic elimination of

619 Tregs due to diminished CA signaling. Conversely, an

620 increased local concentration of CA may be expected to

621 augment the elimination of Tregs. Data indicating that a

622 reduced thymic-dependent de novo generation of recent

623 thymic Tregs may be involved in the pathogenesis of

624 autoimmune diseases [89] point to the putative patho-

625 physiological implications of such a scenario. Moreover,

626 they also impose a question on the relationship between

627 neurally derived and thymocyte/thymic-synthesized CAs.

628 End-point stress mediators and macrophages

629 This section discusses the ability of the end-point stress

630 mediators represented by CAs, NPY, and rodent GC cor-

631 ticosterone, to change the macrophage activity relevant to

632 autoimmune pathology, mainly EAE. It should be pointed

633 out that most data which are conferred have been obtained

634 in studies investigating peritoneal macrophages. This is

635 important in light of data showing that macrophages are

636 cells highly sensitive to properties of the local milieu and

637 that the tissue in which the macrophages reside, together

638with changes in the surrounding environment, shape their

639final phenotype, and determine their functional properties.

640Therefore, some limitations in discussing the findings from

641peritoneal macrophage studies are evident.

642Several lines of evidence indicate the important role of

643macrophages in distinct steps in the pathogenesis of

644inflammatory autoimmune diseases. Their role may be

645illustrated using findings obtained in EAE, where the

646elimination of macrophages in inductive phases of the

647disease prevented the invasion of CNS by the autoreactive

648T-cells [158], whereas inhibition of the macrophage influx

649to CNS delayed the onset and reduced the clinical signs of

650EAE [159]. Although the capacity to produce reactive

651oxygen and nitrogen species directly associates macro-

652phages with the myelin destruction [160], their ability to

653produce NO upon phagocytosis of myelin, which eventu-

654ally leads to T-cell suppression [161], suggests that these

655cells are also implicated in the suppression/resolution of

656EAE. Given that macrophages express MHC II and co-

657stimulatory molecules and possess phagocytic capacity,

658their role is also positioned in the upstream of CNS disease

659progression, at the level of early antigen recognition and

660presentation at the periphery [162] and lymphocyte

661restimulation in brain parenchyma [163].

662Production of reactive oxygen species

663H2O2

664Reactive oxygen species generated primarily by macro-

665phages have been implicated as mediators of demyelination

666and axonal damage in EAE [164]. However, reduced

667ability of the macrophages to generate H2O2 due to an

668impaired NADPH oxidase complex (reflecting mutation of

669gene encoding, one of the components in this complex),

670was associated with severe encephalomyelitis and arthritis

671in mice [165]. This finding, together with our observations

672indicating increased H2O2 production in peritoneal mac-

673rophages from arthritis-resistant AO rats after oil or adju-

674vant administration, suggests a regulatory suppressive

675rather than damaging role of H2O2 [166]. It was also shown

676that immunization with encephalitogen increased already

677high H2O2 production in peritoneal macrophages from the

678EAE-resistant AO strain, but did not change the low level

679of H2O2 production in these cells from DA rats that

680exhibited maximal clinical signs of EAE at the time of

681macrophage harvest [167]. The latter is in line with the lack

682of changes in H2O2 production in peripheral blood mono-

683nuclear cells isolated during clinical EAE from highly

684EAE-susceptible Lewis rats [168]. A high H2O2 production

685level was related to cell apoptosis [169]. This fits in a

686scenario in which the rapid apoptotic clearance of mono-

687nuclear cells infiltrating CNS in EAE contributes to the





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688 resistance of AO rats to the disease induction [170]. On the

689 other hand, data indicate that low oxidative stress in anti-

690 gen-presenting cells favors T-cell activation [171], whereas

691 a low amount of H2O2 in these cells, like in DA rats, favors

692 their survival [172].

693 To decipher the putative effects of stress on macro-

694 phages, we explored the effects of the end-point effector

695 stress mediators on macrophages in vitro and in vivo. In

696 our hands, in vitro non-specific AR agonist arterenol sup-

697 pressed phorbol myristate acetate-stimulated H2O2 pro-

698 duction in macrophages in vitro, whereas propranolol was

699 ineffective in this respect. This suggests an a-AR-mediated

700 CA influence on H2O2 production, which is confirmed by

701 the subsequent findings, indicating that the a1-AR antago-

702 nist ebrantil had the opposite effect [80]. Furthermore, for

703 the first time, we demonstrated the co-expression of a- and

704 b-ARs on cells belonging to a subset of peritoneal mac-

705 rophages [80]. Moreover, the presence of TH and NE was

706 detected in the majority of resident peritoneal macro-

707 phages, indicating CA synthesis by these cells. Collec-

708 tively, the previous findings suggest that NE that is

709 synthesized ectopically in macrophages can modulate

710 H2O2 production in an autocrine/paracrine manner. More-

711 over, it was assumed that macrophage-derived CAs are

712 involved in heterotypic AR regulation, as CAs via b-AR

713 may upregulate a-AR expression [80].

714 In our laboratory, it was also demonstrated that NPY

715 increased peritoneal macrophage peroxide production in

716 vitro through the concomitant activation of Y1 and Y2R

717 [13]. Peripheral blood mononuclear cells also produce

718 NPY [173]. The NPY released from adrenal chromaffin

719 cells regulates the secretion of CA in a paracrine manner

720 [174]. In aggregate, these previous findings have led to the

721 question of whether NPY also influences macrophage

722 activity indirectly through modulation of the ectopic CA

723 release. However, previous exposure of rats to acute psy-

724 chological stress prevented the increase of macrophage

725 H2O2 production induced by a wide range of NPY con-

726 centrations in vitro [16]. Thus, it is likely that by experi-

727 encing action of neuroendocrine mediators released during

728 stress in vivo, the hormonal milieu is changed, thus cre-

729 ating an alteration in the macrophage reactivity to the

730 subsequent challenge in vitro [16].

731 It was noteworthy that GC modulatory action was also

732 dependent on the previous exposure to stress, since a single

733 session of a stressor induced resistance to the effects of

734 dexamethasone, as assessed by the HPA and cytokine

735 responses to lipopolysaccharide [175]. In support of the

736 latter observation are data that in vivo exposure to acute

737 psychological or physical stress paradigms, which compa-

738 rably increase plasma corticosterone concentration [176],

739 prevented an increase in H2O2 production in peritoneal

740 macrophages induced by a low dose of corticosterone in

741vitro, whereas it had no effect on the suppression of H2O2

742production induced by high corticosterone concentrations

743[16]. Therefore, we assume that acute stress leads to a

744functional macrophage resistance to GC action [16]. The

745subsequent research showed that corticosterone in vitro

746exerts stimulatory effects on different macrophage func-

747tions at low concentrations, whereas higher doses are

748mostly suppressive [177]. Furthermore, CD11 ? macro-

749phage resistance to GC action, following chronic social

750stress due to impaired nuclear translocation of GR after GC

751stimulation, and consequent blockade of NF-kappa B

752activation following lasting exposure to increase circulat-

753ing GC levels, has been observed [178]. These findings,

754together with those indicating a long-lasting (24–96 h)

755increase in the level of free biologically active corticoste-

756rone following termination of a single shock session [179],

757suggest that corticosterone increase following acute stress

758is also sufficient to activate macrophage resistance to GC

759action, thereby hindering the potentiating effect of their

760low doses in vitro.

761NO

762NO and its extremely reactive products peroxynitrite and

763nitrotyrosine are formed early in EAE development, and

764their production is linked with clinical disease activity [180].

765NO by its products induces lipid peroxidation and demye-

766lination, but also suppresses lymphocyte proliferation, and

767thus can be viewed as a molecule with a dual role in the

768development and regulation of EAE [161]. Enhanced NO

769production is even considered a possible mechanism of

770resistance to EAE inBrownNorway, PVG, and Fischer F344

771rat strains [181–183]. Immunization with an emulsified

772guinea pig spinal cord did not change peritoneal macrophage

773NO production in DA and AO rats. This suggests that dif-

774ferences in macrophage NO production do not contribute to

775differential susceptibility to EAE in these strains [184].

776Supplying resident peritoneal macrophages with non-

777selective b-AR antagonist propranolol in vitro revealed the

778suppression of NO production [80]. This suggests an

779enhancing b-AR-mediated effect of ectopically synthesized

780CAs on NO production in macrophages. The assumption

781was confirmed by data showing the augmenting effect of

782arterenol on macrophage NO production [80], consistent

783with the ability of b-AR agonist albuterol to induce iNOS

784expression in airway epithelial cells [185]. In contrast,

785chronic treatment with propranolol in vivo enhanced NO

786production by peritoneal macrophages [80]. This is related

787to the increased frequency of b2-AR-expressing peritoneal

788macrophages and the upregulation of b2-AR expression on

789these cells after chronic bAR blockade [80].

790In contrast to the mainly enhancing effect of endogenous

791CAs on macrophage NO production, the effects of NPY on





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792 peritoneal macrophage NO production have been shown to

793 be age-dependent [11, 17]. This may be associated with

794 age-dependent differences in the activity of the NPY-

795 degrading enzyme dipeptidyl-peptidase (DP) 4, which

796 cleaves intact NPY to peptides devoid of Y1R activity. A

797 greater proportion of macrophages expressing the cellular

798 form of enzyme DP4 was also observed in 24-month-old

799 rats, compared to 3- and 8-month-old rats. Moreover,

800 macrophage DP4 activity was higher in aged rats compared

801 with young and adult rats. This implicates that higher DP4

802 activity in aged rats more readily degrades NPY and

803 abolishes its Y1R activity. Considering that Y1 and Y2R

804 have been implicated in the mediation of the potentiating

805 effect of NPY on NO production in young rats, it may be

806 hypothesized that the lack of Y1R signaling in aged rats

807 leads to NPY inefficiency in this respect [11].

808 Moreover, it is noteworthy that there are data that CAs

809 may influence NPY release from hypothalamic neurons

810 [186]. Therefore, it may be speculated that CAs, acting in

811an autocrine/paracrine manner, may influence the release

812of NPY from macrophages and in turn, their action.

813Macrophage NO production was also modulated by

814corticosterone in such a way that low doses increased,

815whereas high concentrations of corticosterone decreased

816NO production [187]. Inactivation of the macrophages’ GR

817by a lentiviral vector bearing small interfering RNA for

818GR abrogated both the immunostimulatory and immuno-

819suppressive GC actions [187]. This suggests that the

820opposing effects of corticosterone on macrophage NO

821production are GR-mediated [188]. Furthermore, it should

822be pointed out that GCs may also influence macrophage

823functions indirectly by affecting CA synthesis [189] and/or

824the expression of ARs in macrophages [190]. Moreover,

825CGs were shown to increase NPY expression [191],

826whereas adrenalectomy was demonstrated to diminish Y1

827and Y5 mRNA expression in hypothalamic neurons [192].

828Considering the aforementioned in this section, a hypo-

829thetical scenario of the end-point stress mediator action on

Fig. 2 Schematic outline of hypothetic interactions between cate-

cholamines (CAs), neuropeptide Y (NPY) and glucocorticoids (GCs)

in the modulation of macrophage functions. The capacity of

macrophages to synthesize and secrete CAs and NPY, along with

the presence of functional adrenergic receptors (ARs) and Y receptors

support the paracrine and autocrine action of macrophage-derived

CAs and NPY. Furthermore, CAs may influence NPY synthesis. GCs

have been shown to influence CA and NPY synthesis and AR and Y

receptor expression. CAs suppress, whereas NPY potentiates the

production of hydrogen peroxide (H2O2) upon macrophage stimula-

tion, acting through a1-AR and Y1/Y2 receptors, respectively (1).

Both CAs and NPY enhance nitric oxide (NO) production from

stimulated macrophages, acting through b-AR and Y1/Y2 receptors,

respectively (2). We hypothesize that b-AR activation facilitates

NPY-induced macrophage NO production, whereas a1-AR activation

abrogates the NPY-induced stimulation of macrophage H2O2 pro-

duction. Moreover, given that chronic b-AR blockade increases a1-

AR expression (through lack of b-AR-mediated stimulatory mecha-

nisms), it may be speculated that chronic blockade of b-ARs may

alter the ultimate functional outcome of macrophage activation by

impairing the b-AR-mediated stimulatory effect of CAs on NO

production and the potentiated a1-AR-mediated suppressive effect of

CAs on H2O2 production





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830 the macrophage production of reactive oxygen species is

831 depicted in Fig. 2.

832 Macrophages as antigen-presenting cells

833 A unique feature of antigen-presenting cells is their ability

834 to recognize and engulf, process and present an antigen to

835 naive T-cells in a MHC II molecule-restricted manner, and

836 to provide the additional signal for T-cells by the expres-

837 sion of co-stimulatory molecules. In addition to dendritic

838 cells, macrophages were also shown to be capable of

839 antigen presentation [162], in which phagocytosis and

840 migration are the first steps. The results of our study

841 revealed that, opposite of peritoneal macrophages from

842 EAE-resistant rats of the AO strain, those from the DA rat

843 strain exhibit a high capacity to phagocyte zymosan. Their

844 phagocytic activity also increases as early as 1 day fol-

845 lowing immunization with encephalitogen [184]. Further-

846 more, there are data indicating that: (1) expression of CD68

847 (ED1) on phagolysosomes correlates with the phagocytic

848 capacity of the cell [193] and (2) increased frequency of

849 CD68 ? cell among peritoneal macrophages 1 day post-

850 immunization with encephalitogen, which may also be

851 related to increased expression of this antigen [184]. Col-

852 lectively, the previous findings suggest a greater ability of

853 peripheral macrophages from DA rats, compared to AO

854 rats, to phagocyte antigen and commence immune activa-

855 tion following immunization. This might be important in

856 light of data showing that, different from AO rats, the

857 influx of CD68 ? macrophages to CNS in DA rats pre-

858 ceded the clonal expansion of T-cells and the activation of

859 microglia [194]. As the expression of CD11b is connected

860 with cell adhesiveness and the ability to transmigrate via

861 lymphatics to the lymph node [195] and infiltrate CNS, the

862 upregulation of CD11b molecule expression on non-resi-

863 dent peritoneal macrophages from DA, but not from AO

864 rats, at an early stage following immunization with

865 encephalitogen [184], probably contributes to the early

866 influx of macrophages to CNS. CD11b is also part of the

867 CD11b/CD18 molecule (Mac-1 or CR3) involved in the

868 phagocytosis of zymosan.

869 Considering that a stress-induced increase in NE is

870 significantly and positively correlated with an increase in

871 the expression of CD11b on human peripheral blood

872 monocytes [196], it might be expected that treatment with

873 CAs affects the phagocytosis of zymosan by macrophages.

874 However, our results reveal that neither the in vitro treat-

875 ment of macrophages with non-specific AR agonist arte-

876 renol or non-selective b-AR antagonist propranolol, nor

877 chronic treatment with propranolol in vivo, have an effect

878 on the phagocytosis of zymosan [80]. In addition, it should

879 be pointed out that Toll-like receptor 2 is also implicated in

880zymosan phagocytosis [197] and that epinephrine down-

881regulates the expression of both toll-like receptor 2 and 4

882on macrophages [198]. Therefore, it is plausible to specu-

883late that CA’s inability to affect zymosan phagocytosis

884reflects its opposing effects on the distinct receptors

885implicated in phagocytosis, that is, CD11b and Toll-like

886receptor 2.

887Concerning NPY, we have found that this mediator

888suppressed the macrophage phagocytosis of zymosan in

889young and adult rats, but not in old rats [13, 16]. This may

890be related to data indicating that: (1) aging is chaperoned

891by an increase in DP4 macrophage activity, which gener-

892ates peptides without Y1R affinity and (2) the suppressive

893effects of NPY and PYY on macrophages are typically

894connected with Y1R signaling.

895It has been shown that acute stress decreases the phag-

896ocytic capacity of murine peritoneal macrophages in a

897corticosterone-dependent way [199]. In line with that our

898results show that the exposure of rats to a psychological

899stressor, as well as in vitro treatment of macrophages with

900corticosterone, suppresses the phagocytosis of zymosan

901[200]. However, previous exposure of the rats to the same

902psychological stress paradigm prevented the suppressive

903effect of corticosterone in vitro, biasing it toward the

904potentiation of phagocytosis [200]. Given that the other

905end-point stress mediators affect the expression of recep-

906tors involved in zymosan phagocytosis [196, 198], it may

907be proposed that flooding the macrophages with different

908mediators released during exposure to the stressor affects

909the expression of receptors for zymosan in such a way that

910subsequent treatment with corticosterone leads to a net

911increase in phagocytosis. In other words, the previous data

912emphasized the hypothesis of active interactions between

913the end-point stress mediators at the macrophage level.

914Conclusions

915In conclusion, although evidence exists that both GCs and

916CAs may affect immunological tolerance at the thymic

917level, it remains elusive how changes (particularly those

918stress-induced) in their action contribute to the triggering

919and development of manifest autoimmune disease. Making

920such projections is particularly difficult, since it has been

921shown that: (1) many of the effects of these mediators on

922the mechanisms of thymic tolerance are, in fact, exerted by

923the mediators ectopically synthesized in the thymus,

924whereas the relationship between extrathymically derived

925and locally synthesized mediators is not yet understood,

926and (2) there is significant crosstalk between GCs and CAs

927not only at the central [96], but also at the thymic level

928[144]. Notwithstanding, we believe that the hereby repor-

929ted outline of potential thymic cellular and molecular





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930 targets of CGs and CAs (Fig. 1) forms a solid scientific

931 basis for further investigation of the putative role of thy-

932 mic-related changes in their action for the pathogenesis of

933 autoimmune diseases.

934 Apart from influencing the mechanisms of central tol-

935 erance at the thymic level, the end-point stress mediators

936 modulate macrophage functions in a strain-, age- and

937 mediator-dependent manner. The final outcome of this

938 modulation depends on the local microenvironment,

939 including adjacent cells and their secretory products, the

940 presence of synthetic or cleaving enzymes, and the com-

941 plex interactions of different CA and NPY receptor sub-

942 types on the macrophage membrane (Fig. 2). In addition,

943 the great variety of roles that macrophages may play in

944 diverse lymphoid and target tissue compartments makes it

945 particularly difficult, but at the same time provocative, to

946 decipher the role of end-point stress mediators in macro-

947 phage biology, and consequently in the pathogenesis of

948 various pathologies, including autoimmune diseases.

949 Acknowledgments We wish to thank our colleagues, Katarina950 Mitic PhD, Vesna Vujic PhD, Vesna Kovacevi-Jovanovic PhD, Du-951 sko Kosec PhD, Katarina Radojevic PhD and Aleksandra Rauski for952 valuable help in the research and stimulating discussion of the results953 that helped us to shape this review. This work was supported by Grant954 (175050) from the Ministry of Education and Science, Belgrade,955 Serbia.

956 References

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End-point effector stress mediators in neuroimmune interactions: their role in immune system...

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