Macrophages, lymphocytes and major histocompatibility complex (HLA) class II antigens in adult human sensory and sympathetic ganglia

Journal of Neuroimmunology, 23 (1989) 187-193

187

Elsevier JNI 00795

Macrophages, lymphocytes and major histocompatibility complex (HLA) class II antigens in adult human sensory and sympathetic ganglia M a r g a r e t M. Esiri a n d M a r g a r e t C. R e a d i n g Neuropathology Department, Radcliffe Infirmary, Oxford OX2 6HE, U.K.

(Received10 August 1988) (Revised, received12 December1988) (Accepted 12 December 1988)

Key words: Macrophage; Lymphocyte;Major histocompatibilitycomplexclass II antigen; Ganglion, human; Immunocytochemistry

Summary We report a n immunocytochernical study of sensory and autonomic ganglia from ten adult human subjects aged 18-83 years without peripheral nerve disease using monoclonal antibodies to macrophages, lymphocytes and human leukocyte (HLA) class II antigens. All ganglia and their associated nerve roots were found to contain a population of resident macrophages which accounted for 5-20% of the cells present. These macrophages and, in addition, many Schwann cells and satellite cells, gave reactions for H L A class II antigens in all cases. Very low numbers of CD3 and CD8 lymphocytes were also regularly detectable in sensory and autonomic ganglia. The resident macrophages may have important immunological and trophic functions. Their possible role in the development of immune-mediated peripheral nerve disease deserves further study.

Introduction

Conventional accounts of the microscopic structure of sensory and sympathetic ganglia describe the principal cellular components as nerve cells and their processes, Schwann cells and satelrite or capsular cells. These lie in an endoneurial stroma containing a few fibroblasts, scattered mast cells and many small blood vessels. The whole structure is ensheathed in a connective tissue capAddress for correspondence: Dr. M.M. Esiri, Neuropathology Department, Radcliffe Infirmary, Oxford OX2 6HE, U.K.

sule continuous with the epineurium of the attached nerve root (Williams and Warwick, 1980; Tennyson and Gershon, 1984). Although there are a few reports of macrophages being found in sensory ganglia (Hamburger and Levi-Montalcini, 1949; Parmese, 1978), these cells are not generally regarded as part of the resident cell population in ganglia. Lymphocytic infiltrates in human sensory ganglia are regarded as a variable finding that may or may not be related to peripheral nerve or ganglion disease. We report here that human sensory and autonomic ganglia contain a substantial population of cells which react immunocytochemically with a panel of macrophage-specific

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188

monoclonal antibodies, have morphological appearances consistent with macrophages and express human leukocyte (HLA) class II antigens. Lymphocytes were found to be a much smaller, less constant and mainly focal constituent of the ganglion cell populations. The regular presence of immune cells in ganglia demonstrated in this study is likely to have implications for the generation of immune-based peripheral nerve diseases.

TABLE 2 MONOCLONAL ANTIBODIES USED IN THE STUDY Antibody

Specificity

Tissue distribution

Reference

EBM/11

NK

Tissue macrophages

Y182a

NK

Tissue macrophages

UCHM1

CD14

RFD7

NK

Tissue macrophages monocytes, dendritic reticulum cells Tissue macrophages

Franklin et al. (1986) Davey et al. (1988) Hogg et al. (1984)

RFD1

NK

T28 T310

CD2 CD4

Tul02

CD8

RFDR1

HLA class II HLA class II

Materials and methods

Samples of sensory (trigeminal and spinal) and autonomic (superior cervical, thoracic and coeliac) ganglia were obtained from ten subjects whose ages ranged from 18 to 83 years (Table 1). Causes of death were: acute head injury (four cases), cerebral glioma (two cases), progressive multifocal leucoencephalopathy complicating Hodgkin's disease (one case), carcinoma of the stomach (one case), congestive cardiac failure (one case) and bronchopneumonia complicating Alzheimer's disease (one case). Neuropathological examination

Mab52

Bct TABLE 1

CD20, CD22

Janossy et al. (1986) Janossy et al. (1986)

Antigen-presenting cells Pan T lymphocytes Helper/inducer lymphocytes, some macrophages Cytotoxic/suppressor lymphocytes Macrophages, B cells, Janossy et activated T cells al. (1986) Macrophages, B cells, Allen and activated T cells Hogg (1987) Pan B cell

SUMMARY OF TEN CASES PROVIDING SAMPLES OF AUTONOMIC AND SENSORY GANGLIA

NK = not known.

Case Sex/ Cause of death No. age (years)

showed no additional incidental abnormalities of the nervous system in any case. Samples were removed at the time of autopsy, embedded in Tissue Tek (Miles) and snap-frozen in liquid nitrogen. Cryostat sections were cut at 6/~m thickness and fixed in acetone at room temperature for 10 min. Sequential sections were chosen for histochemistry to demonstrate acid phosphatase and non-specific esterase activities and immunocytochemistry using a panel of monoclonal antibodies to the following: macrophages (antibodies EBM/11, Y182a, UCHM1 and RFD7), CD3, CD4 and CD8 lymphocytes, HLA class II antigens (antibodies RFDR1 and Mab52) and antigen-presenting cells (antibody RFD1) (Table 2). The neat antibodies were applied to the sections for 45 min at room temperature, the sections were then washed 3 times (5 min each) in Tris-buffered saline, peroxidase-conjugated anti-mouse IgG was

1 2 3

F/18 M/19 M/42

4

M/51

Other disease

Acute head injury Acute head injury Malignant cerebral

glioma 5

6 7 8 9 10

Malignant cerebral glioma M/52 Progressive multifocal Hodgkin's disease leucoencephalopathy in remission with no histological evidence of residual disease at autopsy M/67 Acute head injury A l z h e i m e r ' s disease M/77 Bronchopneumonia Ischaemic M/78 Congestive cardiac myocardial fibrosis failure F/82 Acute head injury F/83 Carcinoma of stomach with. hepatic metastases

189 applied to the sections for 30 min at a dilution of 1 / 5 0 in 1 / 2 0 normal h u m a n serum and after final washing the diaminobenzidine reaction was carded out. Control sections were exposed to normal mouse serum in place of the primary antibody or to an irrelevant antibody (epithelium m e m b r a n e antigen). On sections from selected blocks, combined reactions for acid phosphatase and macrophage-specific antigens were carried out as described previously (Esiri and Reading, 1987).

Results

Macrophages The findings in all cases in sensory and autonomic ganglia were very similar whether or not any central nervous system pathology was present. All ganglia contained a population of cells reactive with the macrophage-specific antibodies. Strong reactions with numerous cells were obtained with antibodies E B M / l l and Y182a and weaker reactions with the antibody U C H M 1 . T h e antibody R F D 7 gave a strong reaction on cells of similar type but reacted with only a small proportion of these cells. Reactive cells were processbearing and variable in shape, usually bipolar, angular or stellate (Fig. 1). They were located in the interstitial tissue between the capsule-bound

Fig. 1. Trigeminal ganglion from case 1. Cryostat-frozen section treated with anti-macrophagemonoelonal antibody Y182a. A positive reaction is seen in the cytoplasm of angular and bipolar cells. The haematoxylin counterstain shows additional nuclei chiefly of satellite cells surrounding the large pale neuron cell bodies CN).x 400.

Fig. 2. Trigeminal ganglion from case 1. Cryostat-frozen section treated with anti-macrophage monoclonal antibody EBMll. Positively reacting cells are seen in close apposition to the neuron cell bodies, lying within the capsules which are chiefly composed of satellite cells (centre). Counterstained with haematoxylin.× 200.

ganglion cells or in the layer of satellite cells composing the capsule. In the latter situation, they were not generally reactive with the R F D 7 antibody and they had an elongated, bipolar form, moulded to the surface of the enclosed ganglion cells (Figs. 2 and 3). Their nuclei were oval, round, kidney shaped or elongated. In haematoxylin and eosin-stained sections, they could not be reliably distinguished from adjacent satellite cells. In rand o m fields in sensory ganglia macrophages accounted for between 10 and 20% of the nucleated cells present depending on the antibody used (Ta-

Fig. 3. Trigeminal ganglion from case 3. Cryostat section treated with anti-macrophage monodonal antibody EBMll. Reactive cells (arrows) lie between the capsules surrounding the neuron cell bodies (N) and within the capsule layer. Haematoxylin counterstain. × 400.

190 TABLE 3 MEAN PERCENTAGES OF NUCLEATED CELLS REACTIVE WITH THREE MACROPHAGE-SPECIFIC MONOCLONAL ANTIBODIES Antibody

EBM/ll Y182a RFD7

Site Sensory ganglion

Sensory nerve root

Superior cervical

Sympathetic chain ganglion

Coeliac plexus ganglion

Nerves of coeliac plexus

20 18 12

18 17 5

19 14 12

18 15 7

16 13 10

16 15 8

Fig. 4. Trigeminal nerve root from case 2. Cryostat section treated with anti-macrophage monoclonal antibody EBMll. Reactive cells appear as elongated bipolar cells lying between the myelinated nerve fibres. The haematoxylin counterstain shows the nuclei of unreactive Schwann cells. × 200.

Fig. 6. Spinal sensory ganglion from case 2. Histochemical reaction for acid phosphatase on a cryostat section. Ganglion nerve cell bodies give a strong reaction (arrows). Capsule cells are unreactive. An interstitial macrophage (arrowhead) gives a weak reaction. × 200.

Fig. 5. Epineural (a) and perivascular (b) macrophages reactive with monoclonal antibody Y182a in the trigeminal ganglion from case 9. Note the elongated form in the epineurium and the plump-bodied form in the perivascular connective tissue. Haematoxylin counterstain. (a) and ( b ) x 400.

Fig. 7. Trigeminal ganglion from case 1. Cryostat section treated with CD3 monoclonal antibody. Scattered CD3 lymphocytes are present (arrows). Counterstained with haematoxylin, x 400.

191 ble 3). The antibody E B M / 1 1 reacted with the largest number of cells, Y182a with slightly fewer cells and R F D 7 with m a n y fewer cells. In sensory nerve roots, 5-18% of nucleated cells reacted with the macrophage-specific antibodies. In this situation, the reactive cells were elongated, slender, bipolar cells aligned adjacent to nerve fibres (Fig. 4), and were indistinguishable from Schwann cells with conventional stains. Cells reactive with macrophage-specific antibodies were also present in the epineurium and in a perivascular location in sensory and autonomic ganglia (Fig. 5). Most of the cells in ganglia and nerve roots that were reactive with macrophage-specific antibodies were unreactive or only very weakly reactive in histochemical reactions for non-specific esterase and acid phosphatase (Fig. 6). In combined reactions for acid phosphatase and the anti-macrophage antibody Y182a, a few doubly reactive cells only were identified in interstitial or perivascular locations. Reactions for antigen-presenting cells using the monoclonal antibody RFD1 were entirely negative.

Lymphocytes All the sensory and autonomic ganglia contained small numbers of cells bearing the CD3 and CD8 marker, but few or no CD4 or B cells were detected. The CD3 and CD8 cells were scattered singly in the interstitial tissue or were located in small clusters around veins (Fig. 7). In

Fig. 9. Small venule in a spinal sensory root from case 3 showing endothelium reactive for HLA class II antigens (Mab52). Counterstalned with haematoxylin.× 400. sensory ganglia, they were more numerous in the trigeminal than the spinal ganglia.

HLA class H antigens Ganglionic macrophages in all cases were reactive for H L A class II antigens. In addition, almost all satellite cells and some Schwann cells were reactive for these antigens (Fig. 8), as were occasional endothelial cells lining small veins (Fig. 9). The reaction in Schwann cells and satellite cells was stronger in sensory than autonomic ganglia. In contrast, neuron cell bodies were, in all cases, unreactive for H L A class II antigens. Control sections showed no positive staining in ganglia or nerve roots.

Discussion

Fig. 8. Tdgerninal ganglion' from case 1. Cryostat section treated with anti-HLA class II antigens (Mab52). A reaction is seen on capsular cells and interstitial macrophages. Neuron cell bodies (N) are negative. Counterstained with haematoxyfin. × 200.

This study has shown that macrophages, immunocytochemically detected using four monoclonal antibodies, are normally resident in adult sensory and autonomic ganglia. There is no clear alteration in the size of this macrophage population with age. In both sensory and autonomic ganglia, the macrophages show a characteristic distribution in the interstitial tissue or adjacent to capsular satellite cells. Their processes sometimes form a component of the capsules that surround the neuron cell bodies. Macrophages are also distributed in nerve roots, epineurium and around blood vessels. Overall, they account for about 5-20% of the cells present in ganglia, the vari-

192 ability depending mainly on the antibody used to detect them. A few show a weak or moderate reaction for acid phosphatase but they are generally unreactive for non-specific esterase. With conventional histological stains they are difficult to distinguish from the more numerous Schwann cells and satellite cells. This population of resident macrophages in peripheral nerve roots and ganglia may be compared with the more specialized mononuclear phagocytes, microglia, found in the central nervous system (Perry and Gordon, 1988). Normal human microglia show a different pattern of immunoreactivity from ganglionic macrophages with the panel of antibodies used in this study, being EBM/ll-positive but not always Y182apositive and RFD7- and UCHMl-negative (Esiri and Reading, 1987). All these antibodi~,~ give a positive reaction with tissue macrophages in nonneural tissues (Hogg et al., 1984; Franklin et al., 1986; Janossy et al., 1986; Davey et al., 1988). In their immunocytochemical reactions with these antibodies, ganglionic macrophages therefore resemble macrophages in non-neural tissues more closely than they resemble normal brain microglia. They also show some histochemical similarity to non-neural tissue macrophages in the possession by some of the cells of acid phosphatase activity, a property that is lost in normal human microglia. In addition to their phagocytic and antigen-presenting roles, resident tissue macrophages are increasingly being recognized to have possible local trophic function (Crocker and Gordon, 1986; Gordon, 1986). In experimental nerve trauma, macrophages have recently been shown to play an essential role in nerve regeneration by producing interleukin-1 which stimulates nerve growth factor production by Schwann cells (Heumann et al., 1987; Lindholm et al., 1987). The close anatomical association of macrophages in ganglia with satellite cells and neurons suggests that they may also have trophic functions to perform normally at this site. An unexpected finding in this study was the strong reaction obtained for HLA class II antigens not only on macrophages but also on some Schwann cells and most satellite cells in sensory and to a lesser extent, autonomic ganglia. In other tissues, these antigens are normally expressed chiefly on macrophages, B cells, activated T cells

and dendritic cells in lymph nodes, but many other cells can be induced to express the antigens under the influence of lymphokines (Klareskog and Forsum, 1986). HLA class II antigen expression by macrophages and dendritic cells is known to be required for T helper cell activation during an immune response to a foreign antigen. It is less clear what part in the generation of local immune responses HLA class II antigen expression by other cells may play and its significance in the case of Schwann cells and satellite cells in ganglia is not known. There is some evidence that expression of HLA class II antigens on resident tissue cells, in contrast to their expression on antigenpresenting cells in lymphoreticular tissues, may have a role in suppressing rather than stimulating local immune responses (Caspi et al., 1987). The absence of any reaction in ganglia with the antibody RFD1 with which antigen-presenting cells in lymph nodes react strongly (Janossy et al., 1986) suggests that antigen presentation by HLA class II-positive cells in ganglia may be modified or suppressed. In comparison with the considerable numbers of macrophages found in ganglia in this study, the numbers of lymphocytes were very low. Nevertheless, CD3 and CD8 cells were regularly found in sensory and autonomic ganglia. It is tempting to speculate over whether these cells carry out a surveillance role in relation to the presence of latent viruses in ganglia, particularly varicella zoster virus and herpes simplex virus. Peripheral infections with both these viruses are more common in the immunosuppressed in whom such a local surveillance role by T lymphocytes might be expected to be suppressed. The trafficking of mononuclear phagocytes and lymphocytes from blood to ganglia, which is implied by the regular presence of these cells in undiseased ganglia, may provide the opportunity for viruses to enter ganglia in infected white blood cells and initiate peripheral nerve and ganglionic disease. Cytomegalovirus, which has been associated with acute inflammatory polyneuropathy (Dowling and Cook, 1981) and human immunodeficiency virus which is associated with several different forms of peripheral neuropathy (Snider et al., 1983; Lipkin et al., 1985; Cornblath et al., 1986; de la Monte, 1988), as well as with

193 g a n g l i o n i t i s ( E l d e r et al., 1986), are e x a m p l e s o f c a n d i d a t e viruses t h a t m a y p e r h a p s g a i n e n t r y to t h e p e r i p h e r a l n e r v o u s s y s t e m in this way.

Acknowledgements T h i s s t u d y r e c e i v e d f i n a n c i a l s u p p o r t f r o m the Medical Research Council. We thank Professor G. Janossy (Royal Free Hospital, London), Professor J. O ' D . M c G e e a n d D r s . D . Y . M a s o n , N . H o g g and K.C. Gatter (John Radcliffe Hospital, Oxf o r d ) for gifts o f a n t i b o d i e s .

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