J .Yi,urr,\i.,crii,r. 199 I . V d . 59, pp. 1-23 Reprints availahlc directly from rhe puhlishcr Photocopying p c r m i l t e d hy license only
lntiw
(
1991 Gordon ;md Breach. Science Puhlijherh S.A.
Printed
in
the IJnited Kingdom
MOLECULAR ANATOMY OF THE NEURO-IMMUNE CONNECTION
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E. WEIHE, D. NOHR, S. MICHEL, S. MULLER, H.-J. ZENTEL, T. FINK and J. KREKEL
Drpcirtnwnt of AnutoniJ*.Johunncs Gutenberg- University, Suurstrasse 19-21, 0-6500 Main,, Gcvwinny Dr. H . - J . Zentel new uddrexs: Scliering A G , Dept. of Phurmucology of tlie Skin, Miillrrstr., D- Ic)OO Berlin, Gertrianv ( ReccJiwd Noiwnihtr.
25, 1990; in final /i,rni 11c~c~t~nihcr. 22, I Y Y O )
Light microscopic immunohistochemistry wits employed to elucidate and compare the presence, distribution. and coexistence of various pcptides, neuroendocrinc markers and enzymes of the catecholamine pathway in nerves supplying lymphoid tissues in a varicty of mammalian species. All lymphoid organs and tissues receive innervation by fibers containing dopamine-/~-hydroxylaseand/or tyrosine hydroxylase, neural markers like protein gene product 9.5. synaptophysin and neurofilament and a varied spectrum of peptides. The prominent peptides were tachykinins (substance P, neurokinin A), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), and vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI). Opioid innervation was variable. Double immunofluorescence revealed coexistence of tachykinins and C G R P and o f tyrosine hydroxylase and NPY. A minor proportion of fibers showed coexisience of NPY and tachykinins and of VIPjPHl and tachykinins. The possible importance of the complex peptidergic innervation of lymphoid tissucs i n inflammation. allergy. inflammatory pain and psycho-neuroimmuno-endocrine network funciion is discussed. A special immunomodulatory role of the sensory neurons is suggested.
Kiyt,ordv: /yn?p/loic/ li.ssuc%, innc~raution. p c ~ ~ ~ r i t itlc~uroinlmunorno~iu~ulutro,l. i~.~, psyclioneuroinlniunol~)~~, .scn.sor ncuron.s 13
The concept that the immune system, the nervous system and the endocrine system are functionally interconnected is gaining support although the detailed mechanisms of this intercommunication in health and disease are not yet clear (Berczi, 1989; Dantzer & Kelly, 1989; Dunn, 1989; Jankovic, 1989; Irwin, Patterson, Smith, Caldwell, Brown, Gillin &Grant, 1990; Weigent, Carr & Blalock, 1990; Weihe, 1990). Two main ways have been suggested through which the nervous system can affect immune responses: an indirect neuroendocrine communication through the secretion of corticosteroids regulated by the hypothalamopituitary-adrenal axis and a direct neural influence on the immune system mediated by the autonomic nervous system (cf, Bateman, Singh, Kral & Solomon, 1989; cf. Bohus & Croiset, 1990). Anatomically, the latter conception is based on the often neglected fact that primary and secondary lymphoid tissues are innervated. The presence of a cholinergic and a sympathetic noradrenergic innervation in lymphoid organs and immunomodulatory influences of the classical cholinergic and noradrenergic transmitters are known for some time (Felten, Felten, Carlson, Olschowka & Livnat, 1985; Kendall, A1 Shawaf & Zaidi, 1988; Lundberg, Hemsen, Rudehill, Hiirfstrand, Larsson, Sollevi, Saria, Hokfelt, The work was supported by the German Research Foundation (We 910/2-1/2-2) and the Stifterverband f u r die Deutsche Wissenschaft (Schering AG). The technical assistance of C . Wigand, S. Bechtloff. B. Ehmer. S. Konrad. and M. Stuhltriger are gratefully acknowledged. Parts of this study will be presented in the thesis of T. Fink and J. Krekel. Send rcprint requests to: Prof. Dr. Eberhard Weihc. Dcpt. of Anatomy, Johannes Gutenberg-Univcrsity. Saarstrasse 19-21. D-6500 Maim, F.R.G.
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2
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Fuxe & Fredholm, 1988; Singh & Fatani, 1988; Ackerman, Felten, Dijkstra, Livnat & Felten, 1989; Madden, Ackerman, Livnat, Felten & Felten, 1989; Van Tits, Michel, Grosse-Wilde, Happcl, Eigler, Soliman & Brodde, 1990). In contrast, the importance of the recently discovered complex autonomic and sensory peptidcrgic innervation of lymphoid tissues in the assumed neuroimmune interplay is unclear. I t appears that all lymphoid organs receive peptidergic innervation (Felten et al. 1985; Fried, Terenius, Brodin, Efendic, Dockray, Fahrenkrug, Goldstein & Hokfelt, 1986; Fink & Weihe, 1988; D’Andrea, Artico, Capuano, Gallotini & Ambrogi, 1989; Weihe, Miiller, Fink & Zentel, 1989; Bellinger, Lorton, Komano, Olschowka, Felten & Felten, 1990; Nohr & Weihe, 1990; Zentel & Weihe, 1990). However, the exact origins, distribution patterns and target relations of peptidergic nerves in the various lymphoid tissues and the possible spectrum of peptides present in such nerves are still incompletely resolved (Kurkowski, Kummer & Heym, 1990; Weihe, 1990). Further, the interrelation of the peptidergic component with noradrenergic and/or cholinergic nerves or with unrecognized nonpeptidic sensory nerves are still unclear (Gibbins, 1990; Weihe, 1990). Many classical transmitters and neuropeptides exert potent immunomodulatory actions in vitro (Costa, Kaylor & Murphy, 1988; Fuchs, Campbell & Munson, 1988; Johansson & Sandberg, 1989; Diilsgaijrd, Hultgardh-Nilsson, Haegerstrand & Nilsson, 1989; Tseng & O’Dorisio, 1989; Umeda & Arisawa, 1989; McGillis, Mitsuhashi & Payan, 1990; Weigent et al. 1990). The in vivo immunomodulatory actions of classical transmitters and particularly of peptides are less well studied (Helme, Eglezos & Hosking, 1987; Fuchs et al. 1988; Moore, Spruck & Said, 1988; Ottaway, 1988; Weigent et al. 1990). Since the presence of neuropeptides in nerves, in conjunction with the occurrence of respective peptide receptors in the various lymphoid target tissues may indicate functional importance, it seems crucial to exactly know which specific compartments of primary and secondary lymphoid tissues do receive peptidergic innervation (Wiedermann, Sertl & Pert, 1986, 1987; Popper, Mantyh, Vigna, Maggio & Mantyh, 1988; Sibinga & Goldstein, 1988; Carr & Blalock, 1989; Weigent et al. 1990). Further, it is obvious that the specific spectrum of peptides present in the nerves supplying the different lymphoid tissues may be decisive for the specific immunomodulatory functions of these nerves. In the light of the coexistence concept of peptidergic innervation (cf. Weihe, 1990) it would be equally important to know about the specific peptide/ peptide(s) and peptide(s)/classical transmitter coexistence patterns in nerves supplying lymphoid tissues. Therefore, it was the aim of this study to address these open questions in providing the missing anatomical comparison and an overview of the peptidergic innervation patterns of the different lymphoid tissues. Particular attention was devoted to elucidate differential neuro-immune target relations and coexistence patterns. Further, the possible implications of the newly recognized details in psycho-neuro-immunoendocrine network functions in allergic. inflammatory, and painful diseases are discussed. MATERIAL AND METHODS Tissue Processing The various lymphoid organs and tissues of several mammalian species were more or less completely investigated as summarized in Table I . Tissues were fixed by immersion and/or perfusion with several fixation solutions, i.e., Bouin’s fluid, Bouin’s fluid withour acetic acid, Rouin de Hollande, Zamboni fixative, 4% phosphate
3
ANATOMY OF THE NEIJRO-IMMUNE CONNECTION TABLE 1 Species and lymphoid tissues investigated
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rat
Y bone marrow thymug Y spleen Y lymph node Y tonsil N BALT Y GALT N NALT Y disseminated lymphoid tissue -gut lamina proprid N -skin subepithelium Y - bronchotrdchedl Y subepithelium
mouse
guine,i-plg
dog
cat
pig
sheep
human
N Y N Y N N N N
Y Y Y Y N Y N N
N N N Y N Y N N
N Y N Y N Y N N
N Y Y N N N N N
N N N Y N N N N
N
N N Y N
N Y N
N Y Y
N Y Y
N Y Y
N Y N
N N N
Y Y N
Y N Y
~
Y : Yes; N: No.
buffered paraformaldehyde solution with or without 1 % glutaraldehyde, and/or nitric acid. Immunocytochemical Procedure und Antisera
Immunoreactions were mostly performed on deparaffinized sections but cryostat sections and free floating sections were also used. A wide spectrum of monoclonal antibodies and polyclonal antisera directed against major opioid and non-opioid peptides, against marker enzymes of the catecholamine pathway, against chromogranin A, and against different markers of the neuroendocrine system, namely synaptophysin, protein gene product 9.5 and neurofilament was employed. For details and abbreviations see Table 2. The immunoreactions were visualized by the use of biotinylated species-specific secondary antisera (Amersham, Sigma, Serva) and the streptavidin-biotin-peroxidasecomplexes (Amersham) reacted with diaminobenzidine (Sigma) and enhanced by ammonium nickel-sulfate (Fluka). For more details of the procedure and for the specificity of the antisera see Miiller and Weihe (1990), Nohr and Weihe (1990), Weihe and Krekel (1990) and Zentel and Weihe (1990). Double Immunofluorescence
Coexistence patterns were evaluated by double-immunofluorescence. Briefly, primary antisera from different species were applied concomitantly. The immunoreactions of the co-applied primary antisera were differentially visualized by using biotinylated antisera and subsequent streptavidin-Texas-Red complex for one antigen and a cascadal system of secondary antisera coupled to FITC to visualize the other antigen. The specificity of the system was carefully assessed as described in more detail elsewhere (Zentel & Weihe, 1990). Co-staining of Immunoreuctive Nerve Fibers and Immune Cells
Neuro-mast cell target relations were determined by successively performing mast cell staining and immune-staining on identical sections. In rats, neuro-macrophage interrelations were determined by co-immunostaining for peptides or neural markers and for the macrophage-related protein ED1 . In human tissues, spatial relationships between immunohistochemically identified nerve fibers and immune cells (B-cells,
E. WElHE 1'1
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TABLE 2 Primary antisera Source
Antigen
Code number
Working dilution
MCA-75S/MAS-O35b
1 :40/1:300 1 :6000 1 : loo00 1 : 1000
,~Ol7-~JpiOit/ /JO/l/id('.S
SP*
Suhstaiice P (pan-tachykinin)
SP SP
Substance P (SP-spccilic) Substance P (SP-specific) Neurokinin A (NKA-specific) Calcitonin gene-related peptide
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NKA C'GRP N PY VIP PHI (;AL** NT CGA CGA CGA**
Ncuropeptide Y Vasoactive intestinal peptide Pcptide histidine isolcucine Galanin Neurotensin Chroniogranin A Chroinogrdnin A Chromogranin A
Op10iti /wp / ides 3E7 pol y-opioid ME-RGL Met-enkephalyl-Arg-Gly-Leu LE Leu-enkephalin DYN A Dynorphin A C'irrc~i~lioliinrrtii~ nicrrhrr
DBH TH**
PGP 9.5
NF** SYN**
-
RAS-7359-N RPN-1842/ RAS-6012-N RPN-I 702 04-340 R-X403 MCA-GFL TH-6 IN 600 86 Lenny LK2 H I 0
I :4000 1 :6000
1:20000 1:20000 1 :500 I :8000 I :4000 I :3000 I:I 1 :60 000 I :20 000 I :30 000 1 : 3000
C. Gramsch E. Weber E. Wcber R. M. Arendt
-
Eugene Tech Boehringer M annheim
T E 103 1017 381
I :50
Serotec
MCA-341
I : 100
Dianova
M 503
I :50
Dakopatts
M 742
I :so
Dakopatts camon
M 754 E-026
1.50 I:50
UltraClone
-
I : 1000
Dianova Boehringer Mannheim
0168 YO2314
1 : 1000 I :200
R-3-3 R- ( - 3 Susi
rn:ynir~.s
Dopamine-/l-hydroxylasc Tyrosinc-hydroxylasc
M a r k o proicim 1 ~ iriiriiuric / c,rll.~ EDI** rat inacrophagcs. monocytes and interdigitating cells LCA** human leucocyte common antigen UCHL I** human antigen-stimulated T cells and memory T cells 4 K B 5** huinaii I3 cells MAC 3X7** human granulocytes, blood iuonocytes and tissue histiocytes Nriiromil
Serotec/Seralab S. Leeman E. Brodin Peninsula Amershani/ Peninsula Amersham CRB N . Yanaihara N . Yanaihara R. Carraway INC L. E. Eiden Histoprime
I :200
riirirker.v
Protein gene product 9.5. ncuronal inarkcr Neurolilament Synaptophysin
* rat monoclonal antibody.
** mouse monoclonal antibody.
T-cells, macrophage cell line, undifferentiated leucocytes) were characterized by successively applying the respective antisera/antibodies for co-immunostaining on identical sections. Neural and immune cell staining was visualized by performing the DAB reaction conventionally or by adding ammonium nickel sulfate resulting in brown and blueish-black reaction products, respectively. For methodological details see Muller and Weihe (l990), Nohr and Weihe (l990), Weihe and Krekel (1990) and Zentel and Weihe ( 1 990).
ANATOMY OF T H E NEURO-IMMUNE CONNECTION
5
RESULTS All fixatives used produced similar results with some marginal differences. The Bouin de Hollande fixative was somewhat superior to the other fixatives.
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Distribution of Immunoreactive Nerve Fibers General patterns As a prominent principal distribution pattern vascular and nonvascular fibers staining for the various peptides and/or markers were present in all lymphoid organs and tissues investigated, with some exceptions exemplified (particularly in the spleen). However, there were some quantitative and qualitative peptide-, marker-, and organ-specific differences (Table 3). As a rule, fibers staining for the pan-neural marker protein PGP 9.5 outnumbered all other immunoreactive (ir) nerve fibers. Only a very minor number of fibers staining for SP or CGRP did not stain for PGP. All fibers staining for PGP stained also for NF, but not all fibers ir for N F were PGP-ir. CGA-immunostaining of some nerve fibers was seen. TH-, NPY-, and VIP/PHI-ir nerve fibers predominantly supplied the vasculature, where they mainly occurred as perivascular plexus. They branched off only rarely to run in the lymphoid parenchyma. In contrast, TK-ir fibers (both SP-ir and NKA-ir) and CGRP-ir fibers regularly branched off from the peri- and paravascular plexus to travel in the respective lymphoid parenchyma. As a striking exception, VIP/PHI-ir fibers were absent from splenic nerves while being present in all other lymphoid tissues investigated as summarized in Table 3 . Opioid immunoreactivity was absent from nerves in the spleen, but present in most other tissues, although in low density. All fibers staining for DBH were also TH-ir, but not all TH-ir fibers contained DBH. DBH immunoreactivity appeared to be restricted to varicose terminals while T H immunoreactivity was also strongly positive in the non-varicose parts of fibers. Synaptophysin immunoreactivity was also restricted to terminals. In contrast, N F TABLE 3 General patterns of vascular and non-vascular distribution and relative frequency of iinmunostained nerve fibers in various lymphoid tissues of different mammalian species PGP vjnv
TH vjnv
NPY vjnv
TK vjnv
CGRP vjnv
OPlOID vjnv
VlPjPHl vjnv
313
Vascular fibers (v). Non-vascular fibers (nv). Subjective rating density of fibers: 0 absent; I very low; 2 low; 3 moderate; 4 high: 5 very high; ? staining equivocal. Bronchus associated lymphoid tissue: BALT. Nasal associated lymphoid tissue: NALT. Gut associated lymphoid tissue: GALT. *in pig only; completely absent in rat and guinea-pig.
6
E. WEIHE
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immunoreactivity prevailed in the non-varicose fiber portions, while PGP immunoreactivity was present in both.
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Bone rnurrow The distribution of ir nerve fibers in the bone marrow could be determined only fragmentarily, because the section and, therefore, the staining quality was unsatisfactory. However, in rat and guinea-pig, CGRP- and tachykinin (TK)-ir fibers were seen to run in vascular and non-vascular locations. Some TH-ir, NPY-ir fibers and NF-ir fibers were also seen. The exact target cells of the non-vascular fibers could not be determined. Thymus The distribution pattern and target relations of ir nerve fibers in the thymus are exemplarily demonstrated in Figures lA, 4C-F, and 6A-B and summarized in Figure 7. The thymic peptidergic innervation predominated in the capsule, in the subcapsular cortex, in interlobular septa and around the vasculature in the cortex and the cortico-medullary boundary. Among the peptide-containing fibers those co-staining for TK and CGRP prevailed (see also Table 3). As analyzed in the rat, close spatial Figure 6B) was relationship with mast cells (Figure 1A) and macrophages (ED1 particularly characteristic for TK/CGRP-ir fibers, but was also seen with some NPY-ir fibers supplying blood vessels (Figure 6A). According to the analysis of adjacent sections, SP- and CGRP-ir nerve fibers completely overlapped, indicating coexistence. This was verified by double immunofluorescence showing co-staining of SP and CGRP in both vascular and nonvascular fibers (Figures 4C-F). In contrast, NPY- and TK/CGRP-immunoreactivities were mostly not co-contained. However, a few NPY-ir fibers also contained ir TKs. Many NPY-ir fibers also stained for TH, but some NPY-ir fibers were TH negative. The possible coexistence of VIP/PHI with NPY or TK/CGRP could not yet be assessed by double immunofluorescence, but the non-coordination pattern indicated that they were mostly non-coexistent. The amount of galanin- and opioid (LE, DYN)-ir nerve fibers was so sparse and variable and the immunosignal so low that possible coexistence patterns could not be assessed yet.
+,
Lymph node Peptide- and marker-ir nerve fibers were concentrated around the vasculature and in the capsular regions. According to the general pattern, TK/CGRPir fibers were the prominent ones to branch off supplying non-vascular areas, particularly in the medullary cord and paracortical (T-cell) regions. As a rule, none of the immunoreactive fibers penetrated into the germinal centers of the lymph follicles. With some predominance to capsular, medullary and cortico-medullary boundary regions TK/CGRP-ir fibers formed close spatial relationship with mast cells (Figure 1 B) and also with macrophages (not shown). Neuro-mast cell contacts also were provided by PGP- and NF-ir fibers (Figure IC). Double immunofluorescence clearly revealed coexistence of SP and CGRP immunoreactivity in vascular and non-vascular fibers (Figure 4A-B) and of TH and NPY in vascular fibers (Figure 5A-B). In contrast, SP and NPY immunoreactivities were mainly non-coexisting. Again, possible coexistence of VIP and PHI could not be assessed. A very few GAL-ir and, at least in guinea-pig, some LE- and DYN-ir fibers were seen. The peptidergic pattern was similar in lymph nodes of different regions. However, there were some quantitative differences, best characterized by a generally lower density of peptidergic fibers in visceral (abdominal) than in cervical (submaxillary, submandibular) or somatic (axillar, popliteal) lymph nodes.
Splecn As mentioned above, the spleen showed a more restricted pattern of peptidergic innervation as compared to the other lymphoid organs and tissues. Note-
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ANATOMY OF T H E NEURO-IMMUNE CONNECTION
FIGURE I LM immunocytochemistry demonstrating close spatial relationship of CGRP-immunoreactive (ir) nerve fibers and neurofilament (NF)-ir nerve fibers with mast cells (arrows) in rat thymus and lymph node. Note that some mast cells are not directly targetted by CGRP-ir nerve fibers. The staining of the mast cells is non-specific. Figure 1A x 280. Figure I B x 280, Figure IC x 275.
E. WEIHE
CI
trl.
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x
FIGURE 2 LM ininiunocytochemistry of pig spleen showing PGP 9.5 (2A)-and tyrosine hydroxylase (TH) (2B)--ir nerve fibers associated with a central artery (a) and a periarteriolar lymphatic sheath. Note high density of PGP 9.5-ir fibers in the red pulp. The nerves in cross and longitudinal profiles are heavily stained for PGP 9.5 and TH respectively (asterisks). TH-ir fibers are present in the periphery of the lymphoid folliclc. i.c.. in the T cell arca (arrowheads). Nerve fibers in the trabecule (t) are strongly TH positive. Figures 2A and B x 300.
9
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ANATOMY OF T H E NEURO-IMMUNE CONNECTION
FIGURE 3 LM immunocytochemistry of mucosa associated lymphoid tissues (MALT). Figure 3A: The gut associated lymphoid tissue (GALT) in human rectum receives PHI-ir innervation in the peripheral part of the GALT only. Note relatively high density of PHI-ir nerve fibers in the surrounding lamina propria beneath the tangentially sectioned rectal crypt epithelium. Figure 3B: CGRP-ir nerve fibers in BALT (bonchus associated lymphoid tissue) of rat lung. Note location of CGRP-ir nerve fibers in the periphery of BALT, but also penetrating towards the center of BALT. CGRP-ir fibers are also present in the zone of smooth muscle (sm) which is partly infiltrated by immune cells. Figure 3A x 310, Figure 3B x 145.
10
E. WEIHE
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crl
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worthy, opioid- and VIP/PHT-ir nerve fibers were absent, in rat and guinea-pig, but present in pig. VR-ir fibers were restricted to the capsule. The PGP-stained fiber population outnumbered the relatively dense and mostly coinciding TH- and NPY-ir subset (Figure 2A-B). In contrast to NPY-ir fibers, PGP-ir fibers were characteristically dense in the red pulp (Figure 2A). In the white pulp, PGP- and TH-ir fibers were concentrated around the pulp artery and the PALS region. Some TH- and PGP-ir fibers accumulated in the outer region of the splenic follicles. As a striking feature, TK/CGRP-ir fibers were sparse (see Table 3). Tonsils Only human tonsils were investigated. The peptidergic innervation was relatively sparse and predominated in vascular association. The peptidergic nerve fibers were clearly outnumbered by those staining for PGP or NF. Neuro-T-cell spatial relationships prevailed, but some neuro-macrophage contacts were also seen. Mucosu-ussociuted lvmphnid tissues ( M A L T ) The innervation of gut-associated lymphoid tissue (GALT) and bronchus-associated lymphoid tissue (BALT) was characteristically concentrated in the peripheral regions of GALT and BALT, respectively, while rarely present in the central parts (Figure 3A-B). In human GALT the target cells of peptidergic innervation were mainly T-cells. In BALT of rats, ED1 macrophages and mast cells were targetted by fibers containing TK/CGRP and ME-RGL (Figure 6C). The CGRP-ir innervation of human GALT was relatively sparse and clearly outnumbered by the TK and VIP/PHI innervation. In fact, we obtained preliminary evidence for a substantial colocalization of VIP/PHI and TK immunoreactivity in nerve fibers supplying the human GALT and the adjacent lamina propria. Interestingly, the density of TK- and VIP/PHI-ir fibers was higher in the lamina propria than in the peripheral GALT (Figure 3A).
+
Coe.xistrnce Patterns
In general, the coexistence patterns followed a unitary scheme. The following pairs of coexistence were characteristic of most lymphoid tissues and species: TH/NPY, TK/CGRP, VlP/PH I . However, there were also some region- and species-specific differences, e.g., a minor portion of NPY-ir fibers of rat thymus also contained ir TK. CGRP immunoreactivity was regularly sparse in the human gastro-intestinal system while the TK and VIP/PHI innervation was equally abundant and often co-distributed indicating co-localization. Spatiul R
lntiw
(
1991 Gordon ;md Breach. Science Puhlijherh S.A.
Printed
in
the IJnited Kingdom
MOLECULAR ANATOMY OF THE NEURO-IMMUNE CONNECTION
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E. WEIHE, D. NOHR, S. MICHEL, S. MULLER, H.-J. ZENTEL, T. FINK and J. KREKEL
Drpcirtnwnt of AnutoniJ*.Johunncs Gutenberg- University, Suurstrasse 19-21, 0-6500 Main,, Gcvwinny Dr. H . - J . Zentel new uddrexs: Scliering A G , Dept. of Phurmucology of tlie Skin, Miillrrstr., D- Ic)OO Berlin, Gertrianv ( ReccJiwd Noiwnihtr.
25, 1990; in final /i,rni 11c~c~t~nihcr. 22, I Y Y O )
Light microscopic immunohistochemistry wits employed to elucidate and compare the presence, distribution. and coexistence of various pcptides, neuroendocrinc markers and enzymes of the catecholamine pathway in nerves supplying lymphoid tissues in a varicty of mammalian species. All lymphoid organs and tissues receive innervation by fibers containing dopamine-/~-hydroxylaseand/or tyrosine hydroxylase, neural markers like protein gene product 9.5. synaptophysin and neurofilament and a varied spectrum of peptides. The prominent peptides were tachykinins (substance P, neurokinin A), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), and vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI). Opioid innervation was variable. Double immunofluorescence revealed coexistence of tachykinins and C G R P and o f tyrosine hydroxylase and NPY. A minor proportion of fibers showed coexisience of NPY and tachykinins and of VIPjPHl and tachykinins. The possible importance of the complex peptidergic innervation of lymphoid tissucs i n inflammation. allergy. inflammatory pain and psycho-neuroimmuno-endocrine network funciion is discussed. A special immunomodulatory role of the sensory neurons is suggested.
Kiyt,ordv: /yn?p/loic/ li.ssuc%, innc~raution. p c ~ ~ ~ r i t itlc~uroinlmunorno~iu~ulutro,l. i~.~, psyclioneuroinlniunol~)~~, .scn.sor ncuron.s 13
The concept that the immune system, the nervous system and the endocrine system are functionally interconnected is gaining support although the detailed mechanisms of this intercommunication in health and disease are not yet clear (Berczi, 1989; Dantzer & Kelly, 1989; Dunn, 1989; Jankovic, 1989; Irwin, Patterson, Smith, Caldwell, Brown, Gillin &Grant, 1990; Weigent, Carr & Blalock, 1990; Weihe, 1990). Two main ways have been suggested through which the nervous system can affect immune responses: an indirect neuroendocrine communication through the secretion of corticosteroids regulated by the hypothalamopituitary-adrenal axis and a direct neural influence on the immune system mediated by the autonomic nervous system (cf, Bateman, Singh, Kral & Solomon, 1989; cf. Bohus & Croiset, 1990). Anatomically, the latter conception is based on the often neglected fact that primary and secondary lymphoid tissues are innervated. The presence of a cholinergic and a sympathetic noradrenergic innervation in lymphoid organs and immunomodulatory influences of the classical cholinergic and noradrenergic transmitters are known for some time (Felten, Felten, Carlson, Olschowka & Livnat, 1985; Kendall, A1 Shawaf & Zaidi, 1988; Lundberg, Hemsen, Rudehill, Hiirfstrand, Larsson, Sollevi, Saria, Hokfelt, The work was supported by the German Research Foundation (We 910/2-1/2-2) and the Stifterverband f u r die Deutsche Wissenschaft (Schering AG). The technical assistance of C . Wigand, S. Bechtloff. B. Ehmer. S. Konrad. and M. Stuhltriger are gratefully acknowledged. Parts of this study will be presented in the thesis of T. Fink and J. Krekel. Send rcprint requests to: Prof. Dr. Eberhard Weihc. Dcpt. of Anatomy, Johannes Gutenberg-Univcrsity. Saarstrasse 19-21. D-6500 Maim, F.R.G.
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2
E. W E I H E
(’I
ti/.
Fuxe & Fredholm, 1988; Singh & Fatani, 1988; Ackerman, Felten, Dijkstra, Livnat & Felten, 1989; Madden, Ackerman, Livnat, Felten & Felten, 1989; Van Tits, Michel, Grosse-Wilde, Happcl, Eigler, Soliman & Brodde, 1990). In contrast, the importance of the recently discovered complex autonomic and sensory peptidcrgic innervation of lymphoid tissues in the assumed neuroimmune interplay is unclear. I t appears that all lymphoid organs receive peptidergic innervation (Felten et al. 1985; Fried, Terenius, Brodin, Efendic, Dockray, Fahrenkrug, Goldstein & Hokfelt, 1986; Fink & Weihe, 1988; D’Andrea, Artico, Capuano, Gallotini & Ambrogi, 1989; Weihe, Miiller, Fink & Zentel, 1989; Bellinger, Lorton, Komano, Olschowka, Felten & Felten, 1990; Nohr & Weihe, 1990; Zentel & Weihe, 1990). However, the exact origins, distribution patterns and target relations of peptidergic nerves in the various lymphoid tissues and the possible spectrum of peptides present in such nerves are still incompletely resolved (Kurkowski, Kummer & Heym, 1990; Weihe, 1990). Further, the interrelation of the peptidergic component with noradrenergic and/or cholinergic nerves or with unrecognized nonpeptidic sensory nerves are still unclear (Gibbins, 1990; Weihe, 1990). Many classical transmitters and neuropeptides exert potent immunomodulatory actions in vitro (Costa, Kaylor & Murphy, 1988; Fuchs, Campbell & Munson, 1988; Johansson & Sandberg, 1989; Diilsgaijrd, Hultgardh-Nilsson, Haegerstrand & Nilsson, 1989; Tseng & O’Dorisio, 1989; Umeda & Arisawa, 1989; McGillis, Mitsuhashi & Payan, 1990; Weigent et al. 1990). The in vivo immunomodulatory actions of classical transmitters and particularly of peptides are less well studied (Helme, Eglezos & Hosking, 1987; Fuchs et al. 1988; Moore, Spruck & Said, 1988; Ottaway, 1988; Weigent et al. 1990). Since the presence of neuropeptides in nerves, in conjunction with the occurrence of respective peptide receptors in the various lymphoid target tissues may indicate functional importance, it seems crucial to exactly know which specific compartments of primary and secondary lymphoid tissues do receive peptidergic innervation (Wiedermann, Sertl & Pert, 1986, 1987; Popper, Mantyh, Vigna, Maggio & Mantyh, 1988; Sibinga & Goldstein, 1988; Carr & Blalock, 1989; Weigent et al. 1990). Further, it is obvious that the specific spectrum of peptides present in the nerves supplying the different lymphoid tissues may be decisive for the specific immunomodulatory functions of these nerves. In the light of the coexistence concept of peptidergic innervation (cf. Weihe, 1990) it would be equally important to know about the specific peptide/ peptide(s) and peptide(s)/classical transmitter coexistence patterns in nerves supplying lymphoid tissues. Therefore, it was the aim of this study to address these open questions in providing the missing anatomical comparison and an overview of the peptidergic innervation patterns of the different lymphoid tissues. Particular attention was devoted to elucidate differential neuro-immune target relations and coexistence patterns. Further, the possible implications of the newly recognized details in psycho-neuro-immunoendocrine network functions in allergic. inflammatory, and painful diseases are discussed. MATERIAL AND METHODS Tissue Processing The various lymphoid organs and tissues of several mammalian species were more or less completely investigated as summarized in Table I . Tissues were fixed by immersion and/or perfusion with several fixation solutions, i.e., Bouin’s fluid, Bouin’s fluid withour acetic acid, Rouin de Hollande, Zamboni fixative, 4% phosphate
3
ANATOMY OF THE NEIJRO-IMMUNE CONNECTION TABLE 1 Species and lymphoid tissues investigated
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rat
Y bone marrow thymug Y spleen Y lymph node Y tonsil N BALT Y GALT N NALT Y disseminated lymphoid tissue -gut lamina proprid N -skin subepithelium Y - bronchotrdchedl Y subepithelium
mouse
guine,i-plg
dog
cat
pig
sheep
human
N Y N Y N N N N
Y Y Y Y N Y N N
N N N Y N Y N N
N Y N Y N Y N N
N Y Y N N N N N
N N N Y N N N N
N
N N Y N
N Y N
N Y Y
N Y Y
N Y Y
N Y N
N N N
Y Y N
Y N Y
~
Y : Yes; N: No.
buffered paraformaldehyde solution with or without 1 % glutaraldehyde, and/or nitric acid. Immunocytochemical Procedure und Antisera
Immunoreactions were mostly performed on deparaffinized sections but cryostat sections and free floating sections were also used. A wide spectrum of monoclonal antibodies and polyclonal antisera directed against major opioid and non-opioid peptides, against marker enzymes of the catecholamine pathway, against chromogranin A, and against different markers of the neuroendocrine system, namely synaptophysin, protein gene product 9.5 and neurofilament was employed. For details and abbreviations see Table 2. The immunoreactions were visualized by the use of biotinylated species-specific secondary antisera (Amersham, Sigma, Serva) and the streptavidin-biotin-peroxidasecomplexes (Amersham) reacted with diaminobenzidine (Sigma) and enhanced by ammonium nickel-sulfate (Fluka). For more details of the procedure and for the specificity of the antisera see Miiller and Weihe (1990), Nohr and Weihe (1990), Weihe and Krekel (1990) and Zentel and Weihe (1990). Double Immunofluorescence
Coexistence patterns were evaluated by double-immunofluorescence. Briefly, primary antisera from different species were applied concomitantly. The immunoreactions of the co-applied primary antisera were differentially visualized by using biotinylated antisera and subsequent streptavidin-Texas-Red complex for one antigen and a cascadal system of secondary antisera coupled to FITC to visualize the other antigen. The specificity of the system was carefully assessed as described in more detail elsewhere (Zentel & Weihe, 1990). Co-staining of Immunoreuctive Nerve Fibers and Immune Cells
Neuro-mast cell target relations were determined by successively performing mast cell staining and immune-staining on identical sections. In rats, neuro-macrophage interrelations were determined by co-immunostaining for peptides or neural markers and for the macrophage-related protein ED1 . In human tissues, spatial relationships between immunohistochemically identified nerve fibers and immune cells (B-cells,
E. WElHE 1'1
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TABLE 2 Primary antisera Source
Antigen
Code number
Working dilution
MCA-75S/MAS-O35b
1 :40/1:300 1 :6000 1 : loo00 1 : 1000
,~Ol7-~JpiOit/ /JO/l/id('.S
SP*
Suhstaiice P (pan-tachykinin)
SP SP
Substance P (SP-spccilic) Substance P (SP-specific) Neurokinin A (NKA-specific) Calcitonin gene-related peptide
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NKA C'GRP N PY VIP PHI (;AL** NT CGA CGA CGA**
Ncuropeptide Y Vasoactive intestinal peptide Pcptide histidine isolcucine Galanin Neurotensin Chroniogranin A Chroinogrdnin A Chromogranin A
Op10iti /wp / ides 3E7 pol y-opioid ME-RGL Met-enkephalyl-Arg-Gly-Leu LE Leu-enkephalin DYN A Dynorphin A C'irrc~i~lioliinrrtii~ nicrrhrr
DBH TH**
PGP 9.5
NF** SYN**
-
RAS-7359-N RPN-1842/ RAS-6012-N RPN-I 702 04-340 R-X403 MCA-GFL TH-6 IN 600 86 Lenny LK2 H I 0
I :4000 1 :6000
1:20000 1:20000 1 :500 I :8000 I :4000 I :3000 I:I 1 :60 000 I :20 000 I :30 000 1 : 3000
C. Gramsch E. Weber E. Wcber R. M. Arendt
-
Eugene Tech Boehringer M annheim
T E 103 1017 381
I :50
Serotec
MCA-341
I : 100
Dianova
M 503
I :50
Dakopatts
M 742
I :so
Dakopatts camon
M 754 E-026
1.50 I:50
UltraClone
-
I : 1000
Dianova Boehringer Mannheim
0168 YO2314
1 : 1000 I :200
R-3-3 R- ( - 3 Susi
rn:ynir~.s
Dopamine-/l-hydroxylasc Tyrosinc-hydroxylasc
M a r k o proicim 1 ~ iriiriiuric / c,rll.~ EDI** rat inacrophagcs. monocytes and interdigitating cells LCA** human leucocyte common antigen UCHL I** human antigen-stimulated T cells and memory T cells 4 K B 5** huinaii I3 cells MAC 3X7** human granulocytes, blood iuonocytes and tissue histiocytes Nriiromil
Serotec/Seralab S. Leeman E. Brodin Peninsula Amershani/ Peninsula Amersham CRB N . Yanaihara N . Yanaihara R. Carraway INC L. E. Eiden Histoprime
I :200
riirirker.v
Protein gene product 9.5. ncuronal inarkcr Neurolilament Synaptophysin
* rat monoclonal antibody.
** mouse monoclonal antibody.
T-cells, macrophage cell line, undifferentiated leucocytes) were characterized by successively applying the respective antisera/antibodies for co-immunostaining on identical sections. Neural and immune cell staining was visualized by performing the DAB reaction conventionally or by adding ammonium nickel sulfate resulting in brown and blueish-black reaction products, respectively. For methodological details see Muller and Weihe (l990), Nohr and Weihe (l990), Weihe and Krekel (1990) and Zentel and Weihe ( 1 990).
ANATOMY OF T H E NEURO-IMMUNE CONNECTION
5
RESULTS All fixatives used produced similar results with some marginal differences. The Bouin de Hollande fixative was somewhat superior to the other fixatives.
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Distribution of Immunoreactive Nerve Fibers General patterns As a prominent principal distribution pattern vascular and nonvascular fibers staining for the various peptides and/or markers were present in all lymphoid organs and tissues investigated, with some exceptions exemplified (particularly in the spleen). However, there were some quantitative and qualitative peptide-, marker-, and organ-specific differences (Table 3). As a rule, fibers staining for the pan-neural marker protein PGP 9.5 outnumbered all other immunoreactive (ir) nerve fibers. Only a very minor number of fibers staining for SP or CGRP did not stain for PGP. All fibers staining for PGP stained also for NF, but not all fibers ir for N F were PGP-ir. CGA-immunostaining of some nerve fibers was seen. TH-, NPY-, and VIP/PHI-ir nerve fibers predominantly supplied the vasculature, where they mainly occurred as perivascular plexus. They branched off only rarely to run in the lymphoid parenchyma. In contrast, TK-ir fibers (both SP-ir and NKA-ir) and CGRP-ir fibers regularly branched off from the peri- and paravascular plexus to travel in the respective lymphoid parenchyma. As a striking exception, VIP/PHI-ir fibers were absent from splenic nerves while being present in all other lymphoid tissues investigated as summarized in Table 3 . Opioid immunoreactivity was absent from nerves in the spleen, but present in most other tissues, although in low density. All fibers staining for DBH were also TH-ir, but not all TH-ir fibers contained DBH. DBH immunoreactivity appeared to be restricted to varicose terminals while T H immunoreactivity was also strongly positive in the non-varicose parts of fibers. Synaptophysin immunoreactivity was also restricted to terminals. In contrast, N F TABLE 3 General patterns of vascular and non-vascular distribution and relative frequency of iinmunostained nerve fibers in various lymphoid tissues of different mammalian species PGP vjnv
TH vjnv
NPY vjnv
TK vjnv
CGRP vjnv
OPlOID vjnv
VlPjPHl vjnv
313
Vascular fibers (v). Non-vascular fibers (nv). Subjective rating density of fibers: 0 absent; I very low; 2 low; 3 moderate; 4 high: 5 very high; ? staining equivocal. Bronchus associated lymphoid tissue: BALT. Nasal associated lymphoid tissue: NALT. Gut associated lymphoid tissue: GALT. *in pig only; completely absent in rat and guinea-pig.
6
E. WEIHE
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immunoreactivity prevailed in the non-varicose fiber portions, while PGP immunoreactivity was present in both.
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Bone rnurrow The distribution of ir nerve fibers in the bone marrow could be determined only fragmentarily, because the section and, therefore, the staining quality was unsatisfactory. However, in rat and guinea-pig, CGRP- and tachykinin (TK)-ir fibers were seen to run in vascular and non-vascular locations. Some TH-ir, NPY-ir fibers and NF-ir fibers were also seen. The exact target cells of the non-vascular fibers could not be determined. Thymus The distribution pattern and target relations of ir nerve fibers in the thymus are exemplarily demonstrated in Figures lA, 4C-F, and 6A-B and summarized in Figure 7. The thymic peptidergic innervation predominated in the capsule, in the subcapsular cortex, in interlobular septa and around the vasculature in the cortex and the cortico-medullary boundary. Among the peptide-containing fibers those co-staining for TK and CGRP prevailed (see also Table 3). As analyzed in the rat, close spatial Figure 6B) was relationship with mast cells (Figure 1A) and macrophages (ED1 particularly characteristic for TK/CGRP-ir fibers, but was also seen with some NPY-ir fibers supplying blood vessels (Figure 6A). According to the analysis of adjacent sections, SP- and CGRP-ir nerve fibers completely overlapped, indicating coexistence. This was verified by double immunofluorescence showing co-staining of SP and CGRP in both vascular and nonvascular fibers (Figures 4C-F). In contrast, NPY- and TK/CGRP-immunoreactivities were mostly not co-contained. However, a few NPY-ir fibers also contained ir TKs. Many NPY-ir fibers also stained for TH, but some NPY-ir fibers were TH negative. The possible coexistence of VIP/PHI with NPY or TK/CGRP could not yet be assessed by double immunofluorescence, but the non-coordination pattern indicated that they were mostly non-coexistent. The amount of galanin- and opioid (LE, DYN)-ir nerve fibers was so sparse and variable and the immunosignal so low that possible coexistence patterns could not be assessed yet.
+,
Lymph node Peptide- and marker-ir nerve fibers were concentrated around the vasculature and in the capsular regions. According to the general pattern, TK/CGRPir fibers were the prominent ones to branch off supplying non-vascular areas, particularly in the medullary cord and paracortical (T-cell) regions. As a rule, none of the immunoreactive fibers penetrated into the germinal centers of the lymph follicles. With some predominance to capsular, medullary and cortico-medullary boundary regions TK/CGRP-ir fibers formed close spatial relationship with mast cells (Figure 1 B) and also with macrophages (not shown). Neuro-mast cell contacts also were provided by PGP- and NF-ir fibers (Figure IC). Double immunofluorescence clearly revealed coexistence of SP and CGRP immunoreactivity in vascular and non-vascular fibers (Figure 4A-B) and of TH and NPY in vascular fibers (Figure 5A-B). In contrast, SP and NPY immunoreactivities were mainly non-coexisting. Again, possible coexistence of VIP and PHI could not be assessed. A very few GAL-ir and, at least in guinea-pig, some LE- and DYN-ir fibers were seen. The peptidergic pattern was similar in lymph nodes of different regions. However, there were some quantitative differences, best characterized by a generally lower density of peptidergic fibers in visceral (abdominal) than in cervical (submaxillary, submandibular) or somatic (axillar, popliteal) lymph nodes.
Splecn As mentioned above, the spleen showed a more restricted pattern of peptidergic innervation as compared to the other lymphoid organs and tissues. Note-
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ANATOMY OF T H E NEURO-IMMUNE CONNECTION
FIGURE I LM immunocytochemistry demonstrating close spatial relationship of CGRP-immunoreactive (ir) nerve fibers and neurofilament (NF)-ir nerve fibers with mast cells (arrows) in rat thymus and lymph node. Note that some mast cells are not directly targetted by CGRP-ir nerve fibers. The staining of the mast cells is non-specific. Figure 1A x 280. Figure I B x 280, Figure IC x 275.
E. WEIHE
CI
trl.
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x
FIGURE 2 LM ininiunocytochemistry of pig spleen showing PGP 9.5 (2A)-and tyrosine hydroxylase (TH) (2B)--ir nerve fibers associated with a central artery (a) and a periarteriolar lymphatic sheath. Note high density of PGP 9.5-ir fibers in the red pulp. The nerves in cross and longitudinal profiles are heavily stained for PGP 9.5 and TH respectively (asterisks). TH-ir fibers are present in the periphery of the lymphoid folliclc. i.c.. in the T cell arca (arrowheads). Nerve fibers in the trabecule (t) are strongly TH positive. Figures 2A and B x 300.
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ANATOMY OF T H E NEURO-IMMUNE CONNECTION
FIGURE 3 LM immunocytochemistry of mucosa associated lymphoid tissues (MALT). Figure 3A: The gut associated lymphoid tissue (GALT) in human rectum receives PHI-ir innervation in the peripheral part of the GALT only. Note relatively high density of PHI-ir nerve fibers in the surrounding lamina propria beneath the tangentially sectioned rectal crypt epithelium. Figure 3B: CGRP-ir nerve fibers in BALT (bonchus associated lymphoid tissue) of rat lung. Note location of CGRP-ir nerve fibers in the periphery of BALT, but also penetrating towards the center of BALT. CGRP-ir fibers are also present in the zone of smooth muscle (sm) which is partly infiltrated by immune cells. Figure 3A x 310, Figure 3B x 145.
10
E. WEIHE
[>f
crl
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worthy, opioid- and VIP/PHT-ir nerve fibers were absent, in rat and guinea-pig, but present in pig. VR-ir fibers were restricted to the capsule. The PGP-stained fiber population outnumbered the relatively dense and mostly coinciding TH- and NPY-ir subset (Figure 2A-B). In contrast to NPY-ir fibers, PGP-ir fibers were characteristically dense in the red pulp (Figure 2A). In the white pulp, PGP- and TH-ir fibers were concentrated around the pulp artery and the PALS region. Some TH- and PGP-ir fibers accumulated in the outer region of the splenic follicles. As a striking feature, TK/CGRP-ir fibers were sparse (see Table 3). Tonsils Only human tonsils were investigated. The peptidergic innervation was relatively sparse and predominated in vascular association. The peptidergic nerve fibers were clearly outnumbered by those staining for PGP or NF. Neuro-T-cell spatial relationships prevailed, but some neuro-macrophage contacts were also seen. Mucosu-ussociuted lvmphnid tissues ( M A L T ) The innervation of gut-associated lymphoid tissue (GALT) and bronchus-associated lymphoid tissue (BALT) was characteristically concentrated in the peripheral regions of GALT and BALT, respectively, while rarely present in the central parts (Figure 3A-B). In human GALT the target cells of peptidergic innervation were mainly T-cells. In BALT of rats, ED1 macrophages and mast cells were targetted by fibers containing TK/CGRP and ME-RGL (Figure 6C). The CGRP-ir innervation of human GALT was relatively sparse and clearly outnumbered by the TK and VIP/PHI innervation. In fact, we obtained preliminary evidence for a substantial colocalization of VIP/PHI and TK immunoreactivity in nerve fibers supplying the human GALT and the adjacent lamina propria. Interestingly, the density of TK- and VIP/PHI-ir fibers was higher in the lamina propria than in the peripheral GALT (Figure 3A).
+
Coe.xistrnce Patterns
In general, the coexistence patterns followed a unitary scheme. The following pairs of coexistence were characteristic of most lymphoid tissues and species: TH/NPY, TK/CGRP, VlP/PH I . However, there were also some region- and species-specific differences, e.g., a minor portion of NPY-ir fibers of rat thymus also contained ir TK. CGRP immunoreactivity was regularly sparse in the human gastro-intestinal system while the TK and VIP/PHI innervation was equally abundant and often co-distributed indicating co-localization. Spatiul R
