Journal of Neuroimmunology, 17 (1988) 315-322 315 Elsevier

JNI 00567

Complement receptors CR1 on human peripheral nerve fibres

C.A. Vedeler and R. Mat re

Broegelmann Research Laboratory for Microbiology and Department of Microbiology and Immunology, The Gade Institute, University of Bergen, Bergen, Norway

(Received 5 June 1987) (Revised, received 4 September 1987)

(Accepted 21 September 1987)

Key words: Complement receptor; Peripheral nerve; Schwann cell

Summary

Receptors for C3 and C4 in human peripheral nerve tissue were studied by examining the adherence of complement (C) coated erythrocytes (E) and by using monoclonal antibodies against epitopes on the receptors for C3b (CR1), C3d (CR2) and C3bi (CR3). E (erythrocyte)-bearing C3b or C4b adhered to sections of myelinated peripheral nerves and the binding was inhibited only by anti-CR1 antibodies. By immunofluorescence, anti-CR1 antibodies stained the nerve fibres, whereas anti-CR2 and anti-CR3 antibodies did not. The staining was apparently localized to the Schwann cell membrane. E-bearing C3bi or C3d did not adhere to myelinated or unmyelinated nerves. CR1 are therefore the only C3 receptors expressed in human peripheral nerves. E-bearing C3b or C4b did not adhere to unmyelinated nerves from adults or to nerves from fetuses at a gestational age of approximately 21 weeks, whereas monoclonal anti-CR1 antibodies stained myelinated, unmyelinated and fetal nerves equally well. The results indicate that CR1 in unmyelinated and fetal nerves are either functionally inactive or express a lower affinity for C3b/C4b than CR1 in myelinated nerves. There were no signifi- cant differences in the binding of E-bearing C3b or C4b to myelinated peripheral nerves from 50 individuals, indicating that CR1 activity is not distributed pheno- typically.

Address for correspondence: C.A. Vedeler, M.D., Broegelmann Research Laboratory for Microbi- ology, MFH-Bygget, Haukeland Hospital, N-5021 Bergen, Norway.

0165-5728/88/$03.50 © 1988 Elsevier Science Pubfishers B.V. (Biomedical Division)

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Introduction

The complement C3b and C4b receptors (CR1) are glycoproteins present on human erythrocytes, neutrophils, lymphocytes and monocytes (Fearon and Wong, 1983) and on podocytes in human renal glomeruli (Matre and Tonder, 1976, 1980). CR1 have also been demonstrated in cryostat sections of myelinated peripheral nerves, by haemadsorption of C3b-coated erythrocytes using a closed chamber technique (Nyland et al., 1979). We have extended our studies on peripheral nerve CR1 to fetal and unmyelinated nerves. We have also used monoclonal antibodies to obtain a more precise localization of CR1. Since the CR1 activity on human erythrocytes (Wilson et al., 1982; Matre and Vedeler, 1987) and in renal glomeruli (Matre et al., 1986/1987) is distributed phenotypically, we have also investigated whether or not phenotypic distribution of the CR1 activity could be demonstrated in human peripheral nerves.

Materials and methods

Tissue

Specimens of human peripheral nerves (oculomotor, olfactory, and sciatic nerves) and renal tissue were obtained at autopsies from the Department of Pathology. The specimens were obtained within 12 h after death from 50 patients (30 males and 20 females, mean age 60 years) who died of cardiac infarction, without signs of neurological or systemic disease. Sciatic nerves were obtained from three human fetuses at a gestational age of approximately 21 weeks. Small pieces of the tissues were frozen in liquid nitrogen and mounted on specimen holders for the preparation of tissue sections. Sections, 4 -6 /~m thick, were cut in a cryostat, placed on covergiasses and air-dried. Histological staining of the tissue specimens with luxol or haematoxylin and eosin revealed their normal structure.

Sera Human sera obtained from healthy blood donors were used as the source of

complement. Human serum heated at 56 °C for 30 min and absorbed with sheep erythrocytes was used as C4b-inactivator reagent as previously described (Matre and Tonder, 1980). Antisera to sheep erythrocytes were produced in rabbits. Mouse monoclonal anti-human CR1 antibody (clone 44D), anti-human CR2 antibody (clone HB5) and anti-human CR3 (clone D12) directed against receptors for C3b, C3d and C3bi respectively, were purchased from Becton Dickinson Laboratory Systems, Belgium. We also used a mouse monoclonal IgG1 antibody against erythrocyte CR1 which was a gift from Dr. Victor Nussenzweig, New York University Medical Center, U.S.A. Rabbit IgG antibodies to mouse IgG conjugated with fluorescein-isothiocyanate (FITC) (code No. F232), FITC-conjugated swine anti-rabbit IgG (code No. F205) and rabbit anti-human C3d (code No. A063) were purchased from DAKO-Immunoglobulins, Copenhagen, Denmark. A mouse mono- clonal antibody, isotyped as IgGlx, against solubilized F c y R from human placenta has been described previously (Matre et al., 1984).

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Immunoglobulins and immune complexes Human IgG (fraction II, 16.5% solution) was purchased from Kabi, Stockholm,

Sweden. Aggregated IgG was obtained by heating the solution at 63°C for 15 min. Immune complexes of horseradish peroxidase (HRP) anti-HRP were prepared as previously described (Matre and Haugen, 1978).

Indicator cells Sheep erythrocytes (E) were sensitized with rabbit antibodies (A) of IgM class

(EA) and incubated in zymosan-treated human serum (C) to obtain EAC3b (Matre and Tonder, 1976). EAC3b were converted to EAC3bi by incubating EAC3b with KCNS-treated serum according to Sandilands and Whaley (1985). To prepare EAC3d, EAC3b were incubated with C3b-inactivator reagent as previously reported (Matre and Tonder, 1976). The indicator cells were made up to a 1% suspension in barbital (Veronal)-buffered saline, pH 7.2, containing 0.15 mM CaCl2, 0.5 mM MgC12 and 0.01% gelatin. In some experiments EAC3b and EAC3bi were sus- pended in Veronal-buffered saline containing 0.1 M EDTA. EAC3bi were also suspended in Veronal-buffered saline containing 2.5% dextrose. EAC3b were posi- tive and EAC3bi and EAC3d were negative in the immune adherence test (Lach- mann and Mfiller-Eberhard, 1968). EAC3b, EAC3bi and EAC3d were agglutinated by rabbit anti-human C3d up to a dilution of I : 1024, indicating that the conversion did not affect the number of C3 molecules on the E. The haemagglutination technique was performed as previously described (Matre and Vedeler, 1977). Ap- proximately 80% of polymorphonuclear (PMN) leucocytes formed rosettes with EAC3b and EAC3bi. EAC3bi did not form rosettes in the presence of EDTA, whereas EAC3b did (Ross et al., 1983). Approximately 10% of peripheral blood mononuclear cells (PBMC) formed rosettes with EAC3d, whereas PMN did not. PMN were prepared according to the method described by Haneberg et al. (1986) and PBMC were obtained by density centrifugation on Lymphoprep (Nyegaard & Co., Oslo, Norway) as described by B~yum (1968). Purity and viability, the latter assessed by exclusion of trypan blue, were > 96%.

EAC4b and EAC4d were prepared as previously described (Matre et al., 1980). Briefly, E were sensitized in unheated rabbit antiserum containing IgG antibodies (A). The EA were then mixed with properly diluted heat-inactivated human serum (C). The mixture was incubated at 37 °C for 15 min and the EAC4b were washed and made up to a 1% suspension in Veronal-buffered saline. To prepare EAC4d, EAC4b were incubated in C4b-inactivator reagent until the cells were unreactive in the immune adherence test.

Haemadsorption to tissue sections for demonstration of CR1 was performed, using the closed chamber technique, as described previously (Matre and Tonder, 1976). The degree of haemadsorption was graded 3 + when the EAC3b/EAC4b adhered to the tissue section in a tight monolayer, 2 + when the section was partially covered by the EAC3b/EAC4b and 1 + when the EAC3b/EAC4b ad- hered as scattered cells. No haemadsorption was recorded as - . In some experi- ments, sections were incubated for 1 h at room temperature with the immune complexes of HRP anti-HRP or with the various monoclonal antibodies diluted 1 : 2

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in phosphate-buffered saline, pH 7.2 (PBS) and washed in PBS before the indicator cells were applied.

Imrnunofluorescence technique The tissue sections were incubated for 18 h at room temperature with the

monoclonal antibodies at various dilutions in PBS. After washing in PBS for 20 rain, the sections were incubated for 1 h with FITC-conjugated rabbit anti-mouse IgG diluted 1 : 30 in PBS containing 25% inactivated pooled human serum (PHS). The sections were then washed in PBS for 20 min and incubated for 1 h with FITC-conjugated swine anti-rabbit IgG diluted 1 :30 in PBS containing 25% in- activated PHS. Controls were prepared by substituting the monoclonal antibodies with PBS or mouse serum. The preparations were washed in PBS, mounted in polyvinylalcohol and examined in a Leitz Orthoplan microscope equipped with an Osram HBO-200 lamp. In some experiments the monoclonal anti-CR1 antibodies were diluted in PBS containing aggregated IgG (4 mg/ml ) to block any binding to FcTR in human peripheral nerves.

R e s u l t s

The EAC3b and EAC4b indicator cells adhered to renal glomeruli and EAC3b adhered to myelinated peripheral nerve fascicles as previously described (Matre and Tonder, 1976, 1980; Nyland et al., 1979). In addition, we found that EAC4b also adhered to myelinated nerves, whereas EAC3b and EAC4b did not adhere to unmyelinated or to fetal nerves (Table 1). EDTA did not prevent the binding of EAC3b or EAC4b, indicating that the binding was not dependent on Ca 2+ and Mg 2+. EAC3bi, EAC3d and EAC4d did not bind to sections of myelinated, unmyelinated and fetal nerves, not even when the indicator cells were suspended in Veronal-buffered saline containing 2.5% dextrose. Taken together, the data indicate that myelinated nerves express only CR1. The same results were obtained with the monoclonal antibodies. The adherence of EAC3b and EAC4b was inhibited by preincubating the sections with monoclonal anti-CR1, but not by monoclonal

TABLE 1

ADSORPTION OF EAC3b/EAC4b PREPARED WITH VARYING AMOUNTS OF HUMAN COM- PLEMENT (C) TO SECTIONS OF HUMAN PERIPHERAL NERVES AND RENAL GLOMERULI

See Materials and Methods for explanation of grading.

Tissue Dilutions of C

1/4 1/8 1/16 1/32 1/64 1 / 1 2 8 1/256 1/512

Myelinated nerves 2 + Unmyelinated nerves - Fetal nerves Renal glomeruli 3 +

1 + 1 + 1 + - - -

3+ 3+ 3+ 2+ 2+ 1+

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ant i -CR2 or ant i -CR3 antibodies. The results also indicate that the adherence of

bo th EAC3b and EAC4b was media ted by CR1. Binding of the monoc lona l ant i -CR1 ant ibodies to peripheral nerves was demon-

strated using a triple-layered immunof luorescence technique. The b i nd i ng was

Fig. 1. Section of sciatic nerve (myelinated) (A) and oculomotor nerve (unmyelinated) (B) incubated with monoclonal anti-CR1 antibody. A: Staining of the outer part of the myelin sheaths (arrows)

( x 400). B: Staining around unmyelinated axons (arrows) ( × 320).

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localized to the nerve fibres, apparently to the Schwann cell membrane (Fig. 1) and the two different" monoclonal anti-CR1 antibodies used gave the same staining pattern. The monoclonal anti-CR1 antibodies from Becton Dickinson Laboratory Systems and from Dr. Victor Nussenzweig stained sections of myelinated, un- myelinated and fetal nerves up to a dilution of 1 : 8 and 1 : 128, respectively. The monoclonal anti-CR2 and anti-CR3 antibodies did not stain peripheral nerves.

The adherence of EAC3b and EAC4b was not inhibited by immune complexes of HRP anti-HRP or by a monoclonal ant i -FcyR antibody. Furthermore, diluting the monoclonal anti-CR1 antibodies in PBS containing aggregated human IgG did not influence the staining pattern or the intensity. These results indicate that the binding was not mediated by F c y R in peripheral nerves (Vedeler, 1987).

In order to obtain binding of EAC3b and EAC4b to peripheral nerves, the indicator cells had to be coated with more C than that required for binding to CR1 in renal glomeruli (Table 1). The results obtained using indicator cells prepared with high concentrations of C as well as the results obtained in titration experiments, did not reveal any significant difference in the degree of haemadsorption to myelinated peripheral nerves from the 50 individuals.

Discussion

Receptors for the activated third component of complement C3b (CR1) have been demonstrated previously in myelinated nerves from individuals aged from 0 to 80 years using EAC3b indicator cells (Nyland et al., 1979). In the present study we demonstrated that EAC4b also adhered to the nerve fascicles of myelinated periph- eral nerves whereas EAC4d did not. The adherence of EAC3b and EAC4b was inhibited by monoclonal anti-CR1, but not by anti-CR2 or anti-CR3 antibodies, indicating that the binding of both EAC3b and EAC4b was mediated by the CR1. We have previously shown that CR1 in human renal glomeruli bind both C3b and C4b (Matre and Tonder, 1980) and Kinoshita et al. (1986) have found that C4b binds to the CR1 on human erythrocytes.

EAC3bi, EAC3d and EAC4d did not bind to peripheral nerves, indicating that peripheral nerves do not express CR2 and CR3. Furthermore, the data indicate that peripheral nerves do not express receptors for C3dg (CR4) as these receptors have been shown to bind both C3bi, C3d and C3dg (Vik and Fearon, 1985). By immunofluorescence, nerve fibres stained with monoclonal anti-CR1 antibodies, but not with anti-CR2 or anti-CR3 antibodies. Thus, by functional and immuno- histochemical criteria, CR1 are the only C3 receptors expressed in human peripheral nerves. Using the same approach, Fischer et al. (1986) showed that CR1 are the only C3 receptors expressed in human glomeruli.

CR1 activity assessed by binding of EAC3b and EAC4b was not detected in unmyelinated nerves or in nerves from fetuses of a gestational age of approximately 21 weeks. This may be due to the necessity for a minimum density of CR1 on the cells in order to allow adherence of EAC3b and EAC4b. However, the monoclonal anti-CR1 antibodies bound equally well to both myelinated, unmyelinated and fetal

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nerves, indicating that approximately the same density of CR1 is present in the nerves. The CR1 in unmyelinated and in fetal nerves may therefore be functionally inactive or express a lower affinity for C3 /C4 compared to the CR1 in myelinated nerves. The results obtained may therefore indicate that functionally active CR1 are related to the production of myelin. That CR1 antigens in peripheral nerve tissue can be detected before functional activity, is of particular interest in relation to the results reported by Appay et al. (1985). They found that CR1 antigens are present in fetal renal glomeruli at a gestational age of approximately 9 weeks, whereas functional CR1 activity was observed only in well-differentiated glomeruli. Whether the same difference in functional CR1 activity can be found also in solubilized form of CR1 from myelinated and unmyelinated nerves is under investigation at present.

The staining pattern of the monoclonal anti-CR1 antibodies indicates that the CR1 are localized to the nerve fibres, apparently to the Schwann cell membrane. However, further studies including electron microscopy have to be performed to determine the exact localization of CR1 in human peripheral nerves.

We could not demonstrate a phenotypic distribution of the CR1 activity in human myelinated peripheral nerves. This is in contrast to the results obtained when the distribution of CR1 on human erythrocytes (Wilson et al., 1982; Matre and Vedeler, 1987) and renal glomeruli (Matre et al., 1986/1987) were studied. However, as the EAC3b and EAC4b bound weakly to myelinated peripheral nerves, the haemadsorption technique may not be appropriate for the demonstration of a possible phenotypic distribution of CR1 in the peripheral nervous system.

In vivo deposits of immunoglobulins and complement have been demonstrated on the nerve fibres in biopsies from patients with the Guillain-Barr6 syndrome (Dalakas and Engel, 1980; Nyland et al., 1981). CR1 may be of significance in the pathogenesis of demyelinating polyneuropathies by trapping C3b-containing im- mune complexes. Furthermore, it has been shown that isolated CR1 from human erythrocytes (Fearon, 1979; Iida and Nussenzweig, 1981) and from renal glomeruli (Fisher et al., 1986) not only bind C3b and C4b, but also function as an important regulator of the classical and alternative pathways by accelerating the decay of C3 convertases and by exhibiting cofactor activity for I-mediated cleavage of C3b and C4b. These functions are of special interest since Koski et al. (1985) have demon- strated that human peripheral myelin can activate complement by the alternative pathway. The CR1 on human peripheral nerve fibres may therefore be of signifi- cance in limiting the damage caused by the complement cascade.

Acknowledgements

We thank Dr. Victor Nussenzweig for the monoclonal anti-CR1 antibody and Mrs. Harriet Aspelund for excellent technical assistance.

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