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Muscarinic cholinergic receptor subtypes in the pigeon bursa of Fabricius: a radioligand binding and autoradiographic study
Journal of Neuroimmunology ELSEVIER
Journal of Neuroimmunology 66 (1996) 23-28
Muscarinic cholinergic receptor subtypes in the pigeon bursa of Fabricius: a radioligand binding and autoradiographic study Albert0
Ricci a, Elena Bronzetti
a’*, Laura Felici a, Emilia Giovanni German& b
Ciriaco
b, Jo& A. Vega ‘,
a Dipartimento di Scienze Cardiovascolari e Kespiratorie, Universita “La Sapienza”, Via A. Borelli 50, 00161 Romn, Italy b Istituto di Anatomia degli Animali Domestici con Istologia ed Embriologia, Universith di Messina. Messina, Italy ’ Departamento de Morfologia y Biologia Cellular, Universidad de Oviedo. Oviedo, Spain
Received 22 August 1995; revised 20 December 1995; accepted 17 January 1996
Abstract The pharmacological profile and the anatomical localisation of muscarinic cholinergic receptor subtypes were studied in the pigeon bursa of Fabricius, using radioligand binding and autoradiographic techniques with [3H]quinuclidinyl benzilate (QNB) as a ligand. [3~]~~~ was specifically bound to sections of bursa of Fabricius. The binding was time-, temperature- and concentration-dependent. The dissociation constant was 0.31 + 0.02 nM, and the maximum density of binding sites averaged 38 f 2.5 fmol/mg protein. The pharmacological profill: of r3H1QNB binding to sections of pigeon bursa of Fabricius was consistent with the labelling of M,, M, and M, muscarinic receptor subtypes. Light microscope autoradiography showed the localisation of t3H]QNB binding sites in the medulla, in follicular septa, in the cortico-medullary border and in lesser amounts in the cortical layer. The functional significance of
these receptors should be clarified in future studies. Keywords: Bursa of Fabricius; M,lscarinic receptor subtypes; Radioligand binding; Autoradiography; Pigeon
1. Introduction
Increasing experimental and clinical evidence suggests the occurrence of interactions between nervous and immune systems (Felten et al., 1985; Ader et al., 1990; Roszman and Brook, 1985). Primary lymphoid organs are supplied by an autonomic -innervation (Zentel et al., 1991; Zentel and Weihe, 1991; Ciriaco et al., 1995; Bellinger et al., 1993) and lymphoid cells express cholinergic, catecholamine and peptide receptors (Hazum et al., 1979; Bishopric et al., 1980; Le Fur et al., 1980; Wybran, 1986; Evans et al., 1986; Maslimki et al., 1990; Takahashi et al., 1992; Nagai et al., 1993; Iiantambrogio et al., 1993; Ricci et al., 1994). The bursa of Fabricius is a unique lymphoid organ which provides the micro-environment for B-lymphocytes (Glick, 1991). The bursa is supplied by peptide-immunoreactive, catecholamine-containing and acetylcholinesterase (AChE)-positive nerve fibres (Zentel et al., 1991; Zentel and Weihe, 1991; Ciriaco et al., 1994), the precise * Corresponding author. Fax i-39 (6) 491 615. 0165-5728/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SOl65-5728(96)00012-4
role of which in modulating its functional activity has not been clarified. Neurotransmitter receptors are expressed by T- and B-lymphocytes of different mammalian species. However, no information is available concerning receptors expressed by the bursa of Fabricius. The bursa of Fabricius is supplied by AChE-containing nerve fibres, probably cholinergic in nature (Ciriaco et al., 1994). In view of this we have investigated the pharmacological profile and the anatomical localisation of muscarinic cholinergic receptor subtypes in the pigeon bursa of Fabricius using combined radioligand binding and autoradiographic techniques.
2. Materials and methods 2.1. Animals
and tissue
treatment
Male king pigeons (Columba livia, L.) (n = 101, 55days-old, were used. They were sacrificed by decapitation under chloral hydrate anaesthesia. The bursae of Fabricius were quickly removed, washed in ice-cold 0.9% NaCl saline solution, embedded in a cryoprotectant medium
24
A. Ricci et al. /Journal
of Neuroimmunology
(OCT, Ames, IA, USA) and frozen in isopentane cooled with liquid nitrogen. OCT blocks were stored at -80°C until used. Serial IO-pm thick sections were obtained using a microtome cryostat and mounted on pre-weighed, gelatine-coated microscope slides. A group of slides for each OCT block was stained with toluidine blue to verify microanatomical details. 2.2. Radioligand
binding experiments
For analysis of muscarinic cholinergic receptors, sections were incubated with increasing concentration of [ 3H]quinuclidinyl benzilate (QNB). Saturation experiments were performed at different times (15, 30,50, 70, 120 min) and temperatures (4”, 25” and 37°C) with radioligand concentrations ranging from 0.1 to 3 nM. Non-specific binding was defined by adding a 1 mM atropine concentration to the incubation medium. The optimal incubation conditions were assessed in a series of preliminary experiments (see Section 3, Results). At the end of incubation, sections were washed twice (2 X 5 min) to remove unbound radioligand and rinsed quickly in distilled water. Sections were then wiped onto Whatman GF-B glass fibre filters and counted by liquid scintillation spectrometry. The pharmacological specificity of [ 3H]QNB binding to sections of pigeon bursa of Fabricius was assessed by incubating some sections with a 0.5 nM [3H]QNB concentration in the presence of increasing concentrations (0.01 nM-20 PM) of compounds active on different subtypes of muscarinic cholinergic receptors. At the end of incubation sections were processed as above. The 0.5 nM [3H]QNB concentration was chosen since it allowed the development of the highest specific/non-specific binding ratio (see Section 3, Results). Representative sections of the bursa of Fabricius of single animals were sonicated and used for total protein determination. 2.3. Light microscope
autoradiography
For light microscope autoradiography, sections of pigeon bursa of Fabricius were incubated with 0.5 nM [3H]QNB in the presence or in the absence of appropriate concentrations of compounds active on muscarinic cholinergic receptor subtypes (see below). After incubation, sections were washed in ice-cold incubation buffer (2 X 5 min), rinsed quickly in distilled water and air-dried. Nuclear emulsion (Ilford L4, diluted 1:l with distilled water)-coated coverslips were then attached to the slides. After exposure for 4-6 weeks in light-tight boxes, autoradiographs were developed in Kodak D19, fixed in Ilford Hypam, stained with toluidine blue and viewed under a bright- and dark-field equipped light microscope. The number of silver grains developed in sections of the pigeon bursa of Fabricius was assessed in dark-field autoradiographs by quantitative image analysis. Measurements were made on five consecutive sections after incubation with 0.5
66 (1996) 23-28
nM i3H]QNB alone, 0.5 nM [3~]~~~ plus 1 /IM atropine to define non-specific binding, or plus 10 PM pirenzepine, 1 PM AFDX 116, 0.1 PM DAMP-methiodide or 0.1 PM tropicamide. Observations for quantitative analysis were made using a Zeiss photomicroscope with a X40/1 .O objective lens and a 2-optovar to obtain a final magnification at the camera of X400. The images were transferred from the microscope to a Quantimet 500 image analyser (Leica, Cambridge, UK) connected via a TV camera to the microscope. Silver grains were counted by image analysis according to the protocol detailed elsewhere (Ricci et al., 1994). 2.4. Data analysis Data from the binding experiments were analysed by linear regression analysis of Scatchard plots of saturation isotherms. Competitor dissociation constant ( Ki) values were determined according to the Cheng and Prussoff equation (Cheng and Prusoff, 1973). Statistical analysis was performed by regression analysis and by analysis of variance (ANOVA). The significance of differences between means was assessed using Duncan’s multiple range test. 2.5. Chemicals [3H]QNB (specific activity 42 Ci/mmol) was purchased from the Amersham Radiochemical Centre (Buckinghamshire, UK). Other compounds were obtained from Research Biomedicals, Inc. (Natick, NJ, USA) or Sigma (St. Louis, MO, USA).
3. Results [3~]QNB binding to sections of pigeon bursa of Fabricius was time-, temperature- (Fig. 1) and concentration-dependent (Fig. 2). Scatchard analysis of saturation isotherms
v-
0
20
40
80
80
100
120
TIME (mid Fig. 1. Influence of different incubation times and temperatures (4”C, m ; 25”C, 0; and 37T. 0) on specific r3H]QNB binding to sections of pigeon bursa of Fabricius. Values are the mean f S.E.M. of 3-5 independent experiments performed as described in Section 2, Materials and methods.
25
A. Ricci et al. /Journal of Neuroimmunology 66 f 1996123-28 Y l”Hl-QNB 6Qecificallybound
ll
10
9
8
7
6
5
4
3
+ + -+
ATR TRO
+
MET
-y-
DAM
+-
ONE
PLP
2
Displacer (-Log Ml Fig. 3. Inhibition of specific [3H]QNB binding to frozen sections of pigeon bursa of Fabricius by increasing concentration of atropine (ATR), quinuclidinyl benzilate (QNB), pirenzepine (PZP), methoctramine (MET), 4-DAMP-methiodide (DAM) and tropicamide (TRO). Sections were incubated with 0.5 nM [ ~H]QNB in the presence of increasing concentrations of compounds tested. Values are the mean k S.E.M. of 3 independent experiments per concentration of displacer performed as indicated in Section 2, Materials and methods.
2s 20 \ 1s 10 S 0; 0.3
6.3
-_‘-.I.-12.3
16.3
.L M--L-. 24.3 30.3
I 3 HbONB BOUND
\
1.6. 36.3
Fig. 2. Upper panel: Saturation curve of [ ‘H]QNB binding to sections of pigeon bursa of Fabricius. Sections were incubated with increasing concentrations of [‘H]QNB alone (total binding: Cl) or plus 1 PM atropine to define non-specific binding (0). Specific binding was determined by subtracting non-specifis from total binding values (m). Points are the meankS.E.M. of 3-5 independent experiments. Lower panel: Scatchard analysis of [3H]QNB binding to sections of pigeon bursa of Fabricius. The B,,, value was 3:s f 2.25 fmol/mg protein.
(Fig. 2) revealed that [ 3H]QNB was bound to a single class of high affinity sites. The dissociation constant value (K,) was 1.23 f 0.04 nM and the maximum binding capacity (B,,,) 38 + 2.5 fmol/mg protein (Fig. 2). Data of pharmacological analysis of [3H]QNB binding to sections of pigeon’s bursa of Fabricius, are shown in Fig. 3 and summarised in Table 1. As can be seen, an-opine was the most powerful competitor of [3H]QNB binding (Fig. 3). Analysis of inhibition constants (Ki), showed the M, receptor subtype antagonist, pirenzepine (Waelbroeck et al., 1990), did have the ability, albeit low, to displace i3H]QNB in sections of pigeon bursa of Fabricius (Fig. 3). Displacement profiles for compounds with an M, receptor
Fig. 4. Light microscope autoracliographs of [‘H]QNB binding to sections of pigeon bursa of Fabricius. Sections were incubated with 0.5 nM [3~]~~ alone (total binding, A) or plus :, PM atropine (non-specific binding, CI. A and C are dark-field pictures. B is a bright-field picture of bursa of Fabricius stained with toluidine blue to verify microanatomical details. c: cortex; is: interfollicular septum; m: medulla. Silver grains sensitive to atropine displacement (specific binding) were accumulated in the medulla followed by interfollicular septa and cortex. (X 105).
26 Table 1 Pharmacological Fabricius
A. Ricci et al. /Journal
specificity
of [3H]QNB
of Neuroimmunology
to sections of pigeon bursa of
Compound
K, (nM)
Receptor specificity
Atropine (+)QNB Pirenzepine AFDXl16 Gallamine Methoctramine DAMP-Methiodide Tropicamide
0.12 & 0.02 3.5 1 & 0.6 220 +12 208 +13 38 f 2.7 7.5 f 0.4 24 f 1.5 5.65 * 0.03
Non-selective muscarinic Non-selective muscarinic M- 1 antagonist M-2 agonist M-2 antagonist M-2 antagonist M-3 antagonist M-4 antagonist
antagonist antagonist
L3HlQNB binding to sections of pigeon bursa of Fabricius was assessed as described in the text. Values represent the competitor dissociation constant ( Ki) determined according to the method of Cheng and Prusoff (1973). Each value is the mean*S.E.M. of 3-5 experiments performed in triplicate. References on the pharmacological specificity of compounds tested are reported in Section 4, Discussion.
66 (1996) 23-28
Table 2 Quantitative analysis of the number of silver grains developed in [3~1~~~ autoradiographs in the different portions of pigeon’s bursa of Fabricius
Total binding Non-specific + PZP +AFDX +DAMP + TROP
Cortex
Medulla
Septa
380&21 70*5a 330+ 15 186* 12 a 234+ 15 b 272*20 b
600+38 105f9 = 560f23 297+15” 375$-21 b 475f32 b
650 f 27 180+ 10 ’ 602 + 33 280f19” 340+ 19b 400*25b
Data are means& S.E.M. and represent the number of silver grains developed in a 500 pm* area of the cortex, of the medulla or of interfollicular septa (septa). Sections were incubated with a 0.5 nM concentration of [ ~H]QNB alone (total binding) or plus 1 PM atropine to define non-specific binding (non-specific). Sections for identifying the muscarinic cholinergic receptor subtypes expressed by different portions of the pigeon bursa of Fabricius were incubated with a 0.5 nM 13HlQNB concentration plus 10 /.t.M pirenzepine (PZP), 1 PM AFDX 116 (AFDX), 0.1 /.tM DAMP-methiodide (DAMP) or 0.1 PM tropicamide (TROP). Details on the technique of silver grains assessment are reported in Section 2, Materials and methods. a P < 0.01 vs. total binding; b P < 0.05 vs. total binding.
Fig. 5. Dark-field autoradiographs of muscarinic cholinergic receptor subtypes in sections of pigeon bursa of Fabricius. Sections were incubated with 0.5 nM [)H]QNB plus 10 pM piretuepine (A) or 1 PM AFDX 116 (B), 0.1 ,uM tropicamide (Cl or 0.1 PM DAMP-methiodide CD). c: cortex, is: interfollicular septum, m: medulla. The lowest density of silver grains was observed in sections incubated with the radioligand plus the Ma receptor agonist AFDX 116 suggesting that the Ma subtype of muscarinic cholinergic receptors is the one most expressed by pigeon’s bursa of Fabricius. (X 105).
subtype selectivity such as gallamine, AFDX 116 and methoctramine were consistent with those typical of muscarinic M, receptor subtype in transfected muscarinic Chinese hamster ovary cells (Buckley et al., 1989). The subtype antagonist, DAMPmuscarinic M 3 receptor methiodide (Buckley et al., 19891, and the muscarinic M, receptor subtype antagonist, tropicamide (Hemandez et al., 19931, displaced [ 3H]QNB with a Ki value of 24 nM and 5.65 nM, respectively (Fig. 3 and Table 1). In light microscope autoradiographs, [ 3H]QNB binding sites appeared as bright silver grains in the dark-field of sections of bursa of Fabricius (Figs. 4 and 5). [3~1~~ specific binding sites (e.g. sensitive to atropine displacement) were found primarily in the medulla and in the cortico-medullary border of pigeon’s bursa of Fabricius. Specific binding was also demonstrated in interfollicular septa (Figs. 4 and 5) and in lesser amounts within the cortical layer (Figs. 4 and 5) and blood vessels of the bursa (data not shown). Quantitative analysis of light microscope autoradiographs performed in the presence of appropriate concentrations of compounds active on different subtypes of muscarinic cholinergic receptors showed that about 60% of specific silver grains was sensitive to displacement by AFDX 116 (Table 2). Specific silver grains sensitive to DAMP-methiodide and tropicamide represent approximately 20% and 10% of specific binding sites, respectively (Fig. 5 and Table 2). The different subtypes of binding sites were located rather homogeneously in portions of the bursa expressing muscarinic cholinergic receptors (Fig. 5 and Table 2).
4. Diiussion The bursa of Fabricius is a bird cloaca1 lymphoepithelial organ in which B-lymphocytes undergo maturation. It
A. Rich et al. / Journal of Neuroimmunology 66 (1996) 23-28
is a diverticulum rich in plicae, located superficially in the body cavity (Ackerman and Knouff, 1959). The plicae entering the organ divide the parenchyma into follicles. Each follicle is anatomically divisible into a cortex and a medulla. The medulla contains reticular cells and fibres, macrophages, plasma cells, lymphoblasts and lymphocytes (Olah and Glick, 1978). The cortex contains lymphoblasts, lymphocytes, macrophages and plasma cells. It is innervated by peptidergic (Felten et al., 1985), noradrenergic (Felten et al., 1985) and AChE-positive (Ciriaco et al., 1995) nerve fibres. The occurrence of a cholinergic modulation of immune function is documented (Maslinski, 1989). The existence of a putative cholinergic innervation of immune organs including the bursa of Fabricius was demonstrated by histochemical, immunocytochemical and ultrastructural techniques (Felten et al., 1985; Ciriaco et al., 1995). AChE-positive nerve fibre:s supply interfollicular areas, cortical blood vessels and the cortico-medullary border (Ciriaco et al., 1995). However, as mentioned in the introduction, no data are available on the presence and localisation of cholinergic receptors in the bursa of Fabricius. Muscarinic cholinergic receptors were demonstrated in immune cells of different species including man (Maslinski et al., 1988). These receptors are structural-related proteins coupled with G proteins (Gilman, 1987; Bonner et al., 1987). Molecular biology techniques have identified 5 different subtypes of muscarinic cholinergic receptors (m 1-m5 sites) cloned from brain and transfected cell lines (Kubo et al., 1986; Perallta et al., 1987; Bonner et al., 1988; Buckley et al., 1989). At present, classic radioligand binding techniques allow one to characterise 4 subtypes of muscarinic cholinergic rea:ptors (M 1-M 4 sites), whereas the m5 receptor subtype generally remains non-characterized by pharmacological methods. Receptors characterised with radioligand binding techniques correspond by number to those identified by molecular biology (Kubo et al., 1986; Peralta et al., 1987; Bonner et al., 1988). The cloned m, receptor demonstrated high affinity for pirenzepine. It represents the M, receptor of the pharmacological classification (Lambrecht et al., 1988; Ehlert et al., 1991). The cloned m2 receptor showed high affinity for gallamine, AFDX 116 and AFDX 382 and methoctramine and is closely related to the M, receptor of classic pharmacology (Lambrecht et al., 1988; Ehlert et al., 1991). The cloned m3 and m4 receptors showed high affinity for 4-DAMP methiodide and tropicamide, respectively, and correspond to M, and M, receptors of classic pharmacology (Michel et al., 1989; Hernandez et al., 1993). Our pharmacological analysis showed that the bursa of Fabricius of adult pigeons expresses muscarinic cholinergic receptors. The affinity of the non-selective muscarinic cholinergic receptor antagonist radioligand [ 3H]QNB and the number of binding slates expressed by the bursa of Fabricius are low in comparison with those of brain or
21
other peripheral organs (De Michele et al., 1989). In spite of this, the development of specific displacement curves in experiments on the pharmacological specificity of [3~]~~ binding suggests that the low concentration of muscarinic cholinergic receptors expressed by the bursa of Fabricius is sufficient to allow their characterization. Muscarinic cholinergic receptors expressed by pigeon bursa of Fabricius are mainly of the M, subtype, with lesser amounts of M, and M, subtypes, whereas M, binding sites (pirenzepine-sensitive) are probably not present in the bursa. This hypothesis is suggested by the high sensitivity of t3H]QNB binding to compounds active on the muscarinic M, receptor subtype, such as AFDX 116, gallamine and methoctramine (Buckley et al., 1989). t3H]QNB binding also displayed sensitivity to compounds active on muscarinic M, and M, receptor subtypes, such as DAMP-methiodide and tropicamide (Buckley et al., 1989; Hemandez et al., 1993) and only slight sensitivity to the muscarinic M, receptor subtype antagonist, pirenzepine (Waelbroeck et al., 1990). Autoradiographic analysis showed that the three muscarinic receptor subtypes expressed by bursa of Fabricius have a similar anatomical localisation. This suggests that targets of muscarinic cholinergic modulation in the bursa of Fabricius are probably the same for the different muscarinic cholinergic receptor subtypes. Muscarinic cholinergic receptors were found both in areas innervated by AChE-positive fibres (Ciriaco et al., 1995) and in non-innervated structures of the bursa of Fabricius. This suggests that muscarinic cholinergic receptors are only in part in relationship with nerve terminals supplying the bursa of Fabricius. Although the functional consequence of this localisation of muscarinic cholinergic receptors cannot be established on the basis of the present study, anatomical localisation suggests their involvement in the modulation of immune cell activity in the bursa of Fabricius. Moreover, in view of the non- absolute selectivity of AChE as a marker of cholinergic innervation (Lehmann and Fibiger, 1979), the demonstration of muscarinic cholinergic receptors in relationship with AChE-positive nerve fibres suggests the occurrence of a cholinergic innervation of the bursa of Fabricius.
Acknowledgements The present study was supported by a grant from the Italian National Research Council (C.N.R.). The authors are greatly indebted to Professor F. Amenta for his critical revision of the manuscript.
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Journal of Neuroimmunology 66 (1996) 23-28
Muscarinic cholinergic receptor subtypes in the pigeon bursa of Fabricius: a radioligand binding and autoradiographic study Albert0
Ricci a, Elena Bronzetti
a’*, Laura Felici a, Emilia Giovanni German& b
Ciriaco
b, Jo& A. Vega ‘,
a Dipartimento di Scienze Cardiovascolari e Kespiratorie, Universita “La Sapienza”, Via A. Borelli 50, 00161 Romn, Italy b Istituto di Anatomia degli Animali Domestici con Istologia ed Embriologia, Universith di Messina. Messina, Italy ’ Departamento de Morfologia y Biologia Cellular, Universidad de Oviedo. Oviedo, Spain
Received 22 August 1995; revised 20 December 1995; accepted 17 January 1996
Abstract The pharmacological profile and the anatomical localisation of muscarinic cholinergic receptor subtypes were studied in the pigeon bursa of Fabricius, using radioligand binding and autoradiographic techniques with [3H]quinuclidinyl benzilate (QNB) as a ligand. [3~]~~~ was specifically bound to sections of bursa of Fabricius. The binding was time-, temperature- and concentration-dependent. The dissociation constant was 0.31 + 0.02 nM, and the maximum density of binding sites averaged 38 f 2.5 fmol/mg protein. The pharmacological profill: of r3H1QNB binding to sections of pigeon bursa of Fabricius was consistent with the labelling of M,, M, and M, muscarinic receptor subtypes. Light microscope autoradiography showed the localisation of t3H]QNB binding sites in the medulla, in follicular septa, in the cortico-medullary border and in lesser amounts in the cortical layer. The functional significance of
these receptors should be clarified in future studies. Keywords: Bursa of Fabricius; M,lscarinic receptor subtypes; Radioligand binding; Autoradiography; Pigeon
1. Introduction
Increasing experimental and clinical evidence suggests the occurrence of interactions between nervous and immune systems (Felten et al., 1985; Ader et al., 1990; Roszman and Brook, 1985). Primary lymphoid organs are supplied by an autonomic -innervation (Zentel et al., 1991; Zentel and Weihe, 1991; Ciriaco et al., 1995; Bellinger et al., 1993) and lymphoid cells express cholinergic, catecholamine and peptide receptors (Hazum et al., 1979; Bishopric et al., 1980; Le Fur et al., 1980; Wybran, 1986; Evans et al., 1986; Maslimki et al., 1990; Takahashi et al., 1992; Nagai et al., 1993; Iiantambrogio et al., 1993; Ricci et al., 1994). The bursa of Fabricius is a unique lymphoid organ which provides the micro-environment for B-lymphocytes (Glick, 1991). The bursa is supplied by peptide-immunoreactive, catecholamine-containing and acetylcholinesterase (AChE)-positive nerve fibres (Zentel et al., 1991; Zentel and Weihe, 1991; Ciriaco et al., 1994), the precise * Corresponding author. Fax i-39 (6) 491 615. 0165-5728/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SOl65-5728(96)00012-4
role of which in modulating its functional activity has not been clarified. Neurotransmitter receptors are expressed by T- and B-lymphocytes of different mammalian species. However, no information is available concerning receptors expressed by the bursa of Fabricius. The bursa of Fabricius is supplied by AChE-containing nerve fibres, probably cholinergic in nature (Ciriaco et al., 1994). In view of this we have investigated the pharmacological profile and the anatomical localisation of muscarinic cholinergic receptor subtypes in the pigeon bursa of Fabricius using combined radioligand binding and autoradiographic techniques.
2. Materials and methods 2.1. Animals
and tissue
treatment
Male king pigeons (Columba livia, L.) (n = 101, 55days-old, were used. They were sacrificed by decapitation under chloral hydrate anaesthesia. The bursae of Fabricius were quickly removed, washed in ice-cold 0.9% NaCl saline solution, embedded in a cryoprotectant medium
24
A. Ricci et al. /Journal
of Neuroimmunology
(OCT, Ames, IA, USA) and frozen in isopentane cooled with liquid nitrogen. OCT blocks were stored at -80°C until used. Serial IO-pm thick sections were obtained using a microtome cryostat and mounted on pre-weighed, gelatine-coated microscope slides. A group of slides for each OCT block was stained with toluidine blue to verify microanatomical details. 2.2. Radioligand
binding experiments
For analysis of muscarinic cholinergic receptors, sections were incubated with increasing concentration of [ 3H]quinuclidinyl benzilate (QNB). Saturation experiments were performed at different times (15, 30,50, 70, 120 min) and temperatures (4”, 25” and 37°C) with radioligand concentrations ranging from 0.1 to 3 nM. Non-specific binding was defined by adding a 1 mM atropine concentration to the incubation medium. The optimal incubation conditions were assessed in a series of preliminary experiments (see Section 3, Results). At the end of incubation, sections were washed twice (2 X 5 min) to remove unbound radioligand and rinsed quickly in distilled water. Sections were then wiped onto Whatman GF-B glass fibre filters and counted by liquid scintillation spectrometry. The pharmacological specificity of [ 3H]QNB binding to sections of pigeon bursa of Fabricius was assessed by incubating some sections with a 0.5 nM [3H]QNB concentration in the presence of increasing concentrations (0.01 nM-20 PM) of compounds active on different subtypes of muscarinic cholinergic receptors. At the end of incubation sections were processed as above. The 0.5 nM [3H]QNB concentration was chosen since it allowed the development of the highest specific/non-specific binding ratio (see Section 3, Results). Representative sections of the bursa of Fabricius of single animals were sonicated and used for total protein determination. 2.3. Light microscope
autoradiography
For light microscope autoradiography, sections of pigeon bursa of Fabricius were incubated with 0.5 nM [3H]QNB in the presence or in the absence of appropriate concentrations of compounds active on muscarinic cholinergic receptor subtypes (see below). After incubation, sections were washed in ice-cold incubation buffer (2 X 5 min), rinsed quickly in distilled water and air-dried. Nuclear emulsion (Ilford L4, diluted 1:l with distilled water)-coated coverslips were then attached to the slides. After exposure for 4-6 weeks in light-tight boxes, autoradiographs were developed in Kodak D19, fixed in Ilford Hypam, stained with toluidine blue and viewed under a bright- and dark-field equipped light microscope. The number of silver grains developed in sections of the pigeon bursa of Fabricius was assessed in dark-field autoradiographs by quantitative image analysis. Measurements were made on five consecutive sections after incubation with 0.5
66 (1996) 23-28
nM i3H]QNB alone, 0.5 nM [3~]~~~ plus 1 /IM atropine to define non-specific binding, or plus 10 PM pirenzepine, 1 PM AFDX 116, 0.1 PM DAMP-methiodide or 0.1 PM tropicamide. Observations for quantitative analysis were made using a Zeiss photomicroscope with a X40/1 .O objective lens and a 2-optovar to obtain a final magnification at the camera of X400. The images were transferred from the microscope to a Quantimet 500 image analyser (Leica, Cambridge, UK) connected via a TV camera to the microscope. Silver grains were counted by image analysis according to the protocol detailed elsewhere (Ricci et al., 1994). 2.4. Data analysis Data from the binding experiments were analysed by linear regression analysis of Scatchard plots of saturation isotherms. Competitor dissociation constant ( Ki) values were determined according to the Cheng and Prussoff equation (Cheng and Prusoff, 1973). Statistical analysis was performed by regression analysis and by analysis of variance (ANOVA). The significance of differences between means was assessed using Duncan’s multiple range test. 2.5. Chemicals [3H]QNB (specific activity 42 Ci/mmol) was purchased from the Amersham Radiochemical Centre (Buckinghamshire, UK). Other compounds were obtained from Research Biomedicals, Inc. (Natick, NJ, USA) or Sigma (St. Louis, MO, USA).
3. Results [3~]QNB binding to sections of pigeon bursa of Fabricius was time-, temperature- (Fig. 1) and concentration-dependent (Fig. 2). Scatchard analysis of saturation isotherms
v-
0
20
40
80
80
100
120
TIME (mid Fig. 1. Influence of different incubation times and temperatures (4”C, m ; 25”C, 0; and 37T. 0) on specific r3H]QNB binding to sections of pigeon bursa of Fabricius. Values are the mean f S.E.M. of 3-5 independent experiments performed as described in Section 2, Materials and methods.
25
A. Ricci et al. /Journal of Neuroimmunology 66 f 1996123-28 Y l”Hl-QNB 6Qecificallybound
ll
10
9
8
7
6
5
4
3
+ + -+
ATR TRO
+
MET
-y-
DAM
+-
ONE
PLP
2
Displacer (-Log Ml Fig. 3. Inhibition of specific [3H]QNB binding to frozen sections of pigeon bursa of Fabricius by increasing concentration of atropine (ATR), quinuclidinyl benzilate (QNB), pirenzepine (PZP), methoctramine (MET), 4-DAMP-methiodide (DAM) and tropicamide (TRO). Sections were incubated with 0.5 nM [ ~H]QNB in the presence of increasing concentrations of compounds tested. Values are the mean k S.E.M. of 3 independent experiments per concentration of displacer performed as indicated in Section 2, Materials and methods.
2s 20 \ 1s 10 S 0; 0.3
6.3
-_‘-.I.-12.3
16.3
.L M--L-. 24.3 30.3
I 3 HbONB BOUND
\
1.6. 36.3
Fig. 2. Upper panel: Saturation curve of [ ‘H]QNB binding to sections of pigeon bursa of Fabricius. Sections were incubated with increasing concentrations of [‘H]QNB alone (total binding: Cl) or plus 1 PM atropine to define non-specific binding (0). Specific binding was determined by subtracting non-specifis from total binding values (m). Points are the meankS.E.M. of 3-5 independent experiments. Lower panel: Scatchard analysis of [3H]QNB binding to sections of pigeon bursa of Fabricius. The B,,, value was 3:s f 2.25 fmol/mg protein.
(Fig. 2) revealed that [ 3H]QNB was bound to a single class of high affinity sites. The dissociation constant value (K,) was 1.23 f 0.04 nM and the maximum binding capacity (B,,,) 38 + 2.5 fmol/mg protein (Fig. 2). Data of pharmacological analysis of [3H]QNB binding to sections of pigeon’s bursa of Fabricius, are shown in Fig. 3 and summarised in Table 1. As can be seen, an-opine was the most powerful competitor of [3H]QNB binding (Fig. 3). Analysis of inhibition constants (Ki), showed the M, receptor subtype antagonist, pirenzepine (Waelbroeck et al., 1990), did have the ability, albeit low, to displace i3H]QNB in sections of pigeon bursa of Fabricius (Fig. 3). Displacement profiles for compounds with an M, receptor
Fig. 4. Light microscope autoracliographs of [‘H]QNB binding to sections of pigeon bursa of Fabricius. Sections were incubated with 0.5 nM [3~]~~ alone (total binding, A) or plus :, PM atropine (non-specific binding, CI. A and C are dark-field pictures. B is a bright-field picture of bursa of Fabricius stained with toluidine blue to verify microanatomical details. c: cortex; is: interfollicular septum; m: medulla. Silver grains sensitive to atropine displacement (specific binding) were accumulated in the medulla followed by interfollicular septa and cortex. (X 105).
26 Table 1 Pharmacological Fabricius
A. Ricci et al. /Journal
specificity
of [3H]QNB
of Neuroimmunology
to sections of pigeon bursa of
Compound
K, (nM)
Receptor specificity
Atropine (+)QNB Pirenzepine AFDXl16 Gallamine Methoctramine DAMP-Methiodide Tropicamide
0.12 & 0.02 3.5 1 & 0.6 220 +12 208 +13 38 f 2.7 7.5 f 0.4 24 f 1.5 5.65 * 0.03
Non-selective muscarinic Non-selective muscarinic M- 1 antagonist M-2 agonist M-2 antagonist M-2 antagonist M-3 antagonist M-4 antagonist
antagonist antagonist
L3HlQNB binding to sections of pigeon bursa of Fabricius was assessed as described in the text. Values represent the competitor dissociation constant ( Ki) determined according to the method of Cheng and Prusoff (1973). Each value is the mean*S.E.M. of 3-5 experiments performed in triplicate. References on the pharmacological specificity of compounds tested are reported in Section 4, Discussion.
66 (1996) 23-28
Table 2 Quantitative analysis of the number of silver grains developed in [3~1~~~ autoradiographs in the different portions of pigeon’s bursa of Fabricius
Total binding Non-specific + PZP +AFDX +DAMP + TROP
Cortex
Medulla
Septa
380&21 70*5a 330+ 15 186* 12 a 234+ 15 b 272*20 b
600+38 105f9 = 560f23 297+15” 375$-21 b 475f32 b
650 f 27 180+ 10 ’ 602 + 33 280f19” 340+ 19b 400*25b
Data are means& S.E.M. and represent the number of silver grains developed in a 500 pm* area of the cortex, of the medulla or of interfollicular septa (septa). Sections were incubated with a 0.5 nM concentration of [ ~H]QNB alone (total binding) or plus 1 PM atropine to define non-specific binding (non-specific). Sections for identifying the muscarinic cholinergic receptor subtypes expressed by different portions of the pigeon bursa of Fabricius were incubated with a 0.5 nM 13HlQNB concentration plus 10 /.t.M pirenzepine (PZP), 1 PM AFDX 116 (AFDX), 0.1 /.tM DAMP-methiodide (DAMP) or 0.1 PM tropicamide (TROP). Details on the technique of silver grains assessment are reported in Section 2, Materials and methods. a P < 0.01 vs. total binding; b P < 0.05 vs. total binding.
Fig. 5. Dark-field autoradiographs of muscarinic cholinergic receptor subtypes in sections of pigeon bursa of Fabricius. Sections were incubated with 0.5 nM [)H]QNB plus 10 pM piretuepine (A) or 1 PM AFDX 116 (B), 0.1 ,uM tropicamide (Cl or 0.1 PM DAMP-methiodide CD). c: cortex, is: interfollicular septum, m: medulla. The lowest density of silver grains was observed in sections incubated with the radioligand plus the Ma receptor agonist AFDX 116 suggesting that the Ma subtype of muscarinic cholinergic receptors is the one most expressed by pigeon’s bursa of Fabricius. (X 105).
subtype selectivity such as gallamine, AFDX 116 and methoctramine were consistent with those typical of muscarinic M, receptor subtype in transfected muscarinic Chinese hamster ovary cells (Buckley et al., 1989). The subtype antagonist, DAMPmuscarinic M 3 receptor methiodide (Buckley et al., 19891, and the muscarinic M, receptor subtype antagonist, tropicamide (Hemandez et al., 19931, displaced [ 3H]QNB with a Ki value of 24 nM and 5.65 nM, respectively (Fig. 3 and Table 1). In light microscope autoradiographs, [ 3H]QNB binding sites appeared as bright silver grains in the dark-field of sections of bursa of Fabricius (Figs. 4 and 5). [3~1~~ specific binding sites (e.g. sensitive to atropine displacement) were found primarily in the medulla and in the cortico-medullary border of pigeon’s bursa of Fabricius. Specific binding was also demonstrated in interfollicular septa (Figs. 4 and 5) and in lesser amounts within the cortical layer (Figs. 4 and 5) and blood vessels of the bursa (data not shown). Quantitative analysis of light microscope autoradiographs performed in the presence of appropriate concentrations of compounds active on different subtypes of muscarinic cholinergic receptors showed that about 60% of specific silver grains was sensitive to displacement by AFDX 116 (Table 2). Specific silver grains sensitive to DAMP-methiodide and tropicamide represent approximately 20% and 10% of specific binding sites, respectively (Fig. 5 and Table 2). The different subtypes of binding sites were located rather homogeneously in portions of the bursa expressing muscarinic cholinergic receptors (Fig. 5 and Table 2).
4. Diiussion The bursa of Fabricius is a bird cloaca1 lymphoepithelial organ in which B-lymphocytes undergo maturation. It
A. Rich et al. / Journal of Neuroimmunology 66 (1996) 23-28
is a diverticulum rich in plicae, located superficially in the body cavity (Ackerman and Knouff, 1959). The plicae entering the organ divide the parenchyma into follicles. Each follicle is anatomically divisible into a cortex and a medulla. The medulla contains reticular cells and fibres, macrophages, plasma cells, lymphoblasts and lymphocytes (Olah and Glick, 1978). The cortex contains lymphoblasts, lymphocytes, macrophages and plasma cells. It is innervated by peptidergic (Felten et al., 1985), noradrenergic (Felten et al., 1985) and AChE-positive (Ciriaco et al., 1995) nerve fibres. The occurrence of a cholinergic modulation of immune function is documented (Maslinski, 1989). The existence of a putative cholinergic innervation of immune organs including the bursa of Fabricius was demonstrated by histochemical, immunocytochemical and ultrastructural techniques (Felten et al., 1985; Ciriaco et al., 1995). AChE-positive nerve fibre:s supply interfollicular areas, cortical blood vessels and the cortico-medullary border (Ciriaco et al., 1995). However, as mentioned in the introduction, no data are available on the presence and localisation of cholinergic receptors in the bursa of Fabricius. Muscarinic cholinergic receptors were demonstrated in immune cells of different species including man (Maslinski et al., 1988). These receptors are structural-related proteins coupled with G proteins (Gilman, 1987; Bonner et al., 1987). Molecular biology techniques have identified 5 different subtypes of muscarinic cholinergic receptors (m 1-m5 sites) cloned from brain and transfected cell lines (Kubo et al., 1986; Perallta et al., 1987; Bonner et al., 1988; Buckley et al., 1989). At present, classic radioligand binding techniques allow one to characterise 4 subtypes of muscarinic cholinergic rea:ptors (M 1-M 4 sites), whereas the m5 receptor subtype generally remains non-characterized by pharmacological methods. Receptors characterised with radioligand binding techniques correspond by number to those identified by molecular biology (Kubo et al., 1986; Peralta et al., 1987; Bonner et al., 1988). The cloned m, receptor demonstrated high affinity for pirenzepine. It represents the M, receptor of the pharmacological classification (Lambrecht et al., 1988; Ehlert et al., 1991). The cloned m2 receptor showed high affinity for gallamine, AFDX 116 and AFDX 382 and methoctramine and is closely related to the M, receptor of classic pharmacology (Lambrecht et al., 1988; Ehlert et al., 1991). The cloned m3 and m4 receptors showed high affinity for 4-DAMP methiodide and tropicamide, respectively, and correspond to M, and M, receptors of classic pharmacology (Michel et al., 1989; Hernandez et al., 1993). Our pharmacological analysis showed that the bursa of Fabricius of adult pigeons expresses muscarinic cholinergic receptors. The affinity of the non-selective muscarinic cholinergic receptor antagonist radioligand [ 3H]QNB and the number of binding slates expressed by the bursa of Fabricius are low in comparison with those of brain or
21
other peripheral organs (De Michele et al., 1989). In spite of this, the development of specific displacement curves in experiments on the pharmacological specificity of [3~]~~ binding suggests that the low concentration of muscarinic cholinergic receptors expressed by the bursa of Fabricius is sufficient to allow their characterization. Muscarinic cholinergic receptors expressed by pigeon bursa of Fabricius are mainly of the M, subtype, with lesser amounts of M, and M, subtypes, whereas M, binding sites (pirenzepine-sensitive) are probably not present in the bursa. This hypothesis is suggested by the high sensitivity of t3H]QNB binding to compounds active on the muscarinic M, receptor subtype, such as AFDX 116, gallamine and methoctramine (Buckley et al., 1989). t3H]QNB binding also displayed sensitivity to compounds active on muscarinic M, and M, receptor subtypes, such as DAMP-methiodide and tropicamide (Buckley et al., 1989; Hemandez et al., 1993) and only slight sensitivity to the muscarinic M, receptor subtype antagonist, pirenzepine (Waelbroeck et al., 1990). Autoradiographic analysis showed that the three muscarinic receptor subtypes expressed by bursa of Fabricius have a similar anatomical localisation. This suggests that targets of muscarinic cholinergic modulation in the bursa of Fabricius are probably the same for the different muscarinic cholinergic receptor subtypes. Muscarinic cholinergic receptors were found both in areas innervated by AChE-positive fibres (Ciriaco et al., 1995) and in non-innervated structures of the bursa of Fabricius. This suggests that muscarinic cholinergic receptors are only in part in relationship with nerve terminals supplying the bursa of Fabricius. Although the functional consequence of this localisation of muscarinic cholinergic receptors cannot be established on the basis of the present study, anatomical localisation suggests their involvement in the modulation of immune cell activity in the bursa of Fabricius. Moreover, in view of the non- absolute selectivity of AChE as a marker of cholinergic innervation (Lehmann and Fibiger, 1979), the demonstration of muscarinic cholinergic receptors in relationship with AChE-positive nerve fibres suggests the occurrence of a cholinergic innervation of the bursa of Fabricius.
Acknowledgements The present study was supported by a grant from the Italian National Research Council (C.N.R.). The authors are greatly indebted to Professor F. Amenta for his critical revision of the manuscript.
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