Human microglial cells have phenotypic and functional characteristics in common with both macrophages and dendritic antigen-presenting cells.

Resting microglia comprise up to 13% of the cells in human central nervous system (CNS) white matter. Their large number and dendritic morphology make them ideally suited to survey the CNS for noxious stimuli. Upon activation microglia gradually lose dendritic processes and transform into typical phagocytic macrophages. Microglia have been implicated as the main antigen presenting cell within the CNS, and appear to be of central importance as effectors and regulators of demyelination. To further characterize the capacity for immune reactivity within the human CNS, we have studied several characteristics of microglia, both in situ and in vitro. We find that human microglia have ultrastructural, phenotypic (CD11c, CD68, acid phosphatase), and functional (FcR and CR mediated phagocytosis) properties typical for cells of the monocyte lineage. Our data indicate that microglia also have properties in common with dendritic antigen-presenting cells. Electron microscopy studies show extended dendritic cell processes on cultured microglia, and microglia are, like dendritic cells, negative for the monocyte markers nonspecific esterase, endogenous peroxidase, CD14, and RFD7. Microglia constitutively express HLA-DR in situ, and express the dendritic cell marker RFD1 upon activation. Coculturing of microglia with CD4+ T cells results in clustering of T cells around microglia and initiation of a mixed lymphocyte reaction, both distinguishing features of dendritic cells. These functional properties of microglia may be of importance for the maintenance of an immunologic response in the CNS, an organ where dendritic cells, in contrast to other organs, have not previously been identified.

Characterization of mononuclear cell infiltrates in psoriatic lesions.

The dermal mononuclear cell infiltrates of psoriatic lesions were characterized by receptors for sheep erythrocytes (T-lymphocytes), C3b receptors (macrophages and B-lymphocytes) and C3d receptors (B-lymphocytes), using hemadsorption to cryostat sections in a closed chamber. T-lymphocytes and macrophages were the predominant inflammatory cells. Very few B-lymphocytes were detected. These findings are discussed in relation to the possible pathogenetic signficance of cellular immunity in psoriasis.

Fc receptors for IgG on cultured human microglia mediate cytotoxicity and phagocytosis of antibody-coated targets.

We have utilized surgically resected human central nervous system (CNS) tissue to determine the expression and functions of Fc receptors (Fc gamma R) on individual cell types found within the CNS. We observed all three classes of Fc gamma R on microglial cells in situ and in vitro, but not on astrocytes or oligodendrocytes. Incubation of cultured microglia with immune complexes (antibody-coated red blood cells) induced phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and oxidative bursts. We also found that microglia have the capability to produce T cell stimulatory soluble mediators after Fc gamma R crosslinking. These functional responses were enhanced by pretreatment of the microglia with interferon-gamma (IFN-gamma). Our results implicate microglial effector responses triggered by interaction of Fc gamma R with opsonized antigens as potential mediators of tissue injury within the CNS. Such injury may be particularly applicable to multiple sclerosis, an inflammatory demyelinating disease characterized by intrathecal production of immunoglobulins and cytokines.

Myasthenia gravis: difference between thymoma-associated antibodies and cross-striational skeletal muscle antibodies.

Serum from 20 of 137 patients (15%) with myasthenia gravis (MG) contained antibodies to a citric acid extract (CAE) of skeletal muscle. A thymoma was found in all 19 of these patients who were thymectomized. There was no evidence of thymoma in patients who did not have CAE antibodies. Serum from 40 of the same 137 patients (30%) contained antibodies that gave a cross-striational immunofluorescent pattern in skeletal muscle. This pattern was seen with serum from 16 of the 20 thymoma patients, but also with serum from 24 patients without thymoma. The cross-striation antibodies were not absorbed by CAE coated on to tanned sheep erythrocytes. Accordingly, antibodies detected in the two tests have different specificities.

An improved method for the detection of human T lymphocytes in tissue sections.

The technique of haemadsorption to tissue sections for the detection of T lymphocytes is described in detail. The modification using the closed chamber technique is convenient, sensitive and reproducible. In principle, it parallels the rosette technique using blood lymphocytes in suspensions and AET-treated sheep erythrocytes. Storage of tissue at -25 degrees C did not reduce the activity of the E-receptors in the tissue. The E-receptors were blocked by incubation of the tissue sections with an anti-T lymphocyte serum.

A simplified method for the preparation of EAC14.

Sheep erythrocytes (E), sensitized with unheated rabbit antiserum containing IgG antibodies (EA), bound human C4 when heat-inactivated serum was used as the source of complement (EAC). The EA were not agglutinated by antiserum to rabbit C3 and were not lysed in the presence of C4-deficient guinea pig serum. The EAC were agglutinated by antiserum to human C4, lysed by the addition of C4-deficient serum and were reactive in immune adherence. They were not agglutinated by antiserum to human C3. Accordingly, the cells could be designated as EAC14. They were stable and could be stored at 4 degrees C for at least 1 week without haemolysis occurring, and with no loss of reactivity in immune adherence test. E sensitized in the presence of EDTA or with heat-inactivated rabbit serum did not bind C4.

Demonstration of human erythrocyte C3b receptors (CR1) by haemadsorption and indirect haemagglutination techniques.

A closed chamber haemadsorption (HA) technique with tissue sections and an indirect haemagglutination (IHA) technique with monoclonal anti-C3b receptor (CR1) antibody were used to measure CR1 on human erythrocytes (E) (ECR1). In the HA technique the indicator cells were sheep E coated with rabbit IgM antibodies (A) and various amounts of human C3b (EAC). The ECR1 activity was classified according to the degree of haemadsorption of EAC. High ECR1 activity was found in 24% of healthy blood donors, intermediate activity in 62%, low activity in 12% and no activity in 2% of the blood donors. The results obtained using the IHA technique showed a good correlation with the results obtained using the HA technique. Both techniques are sensitive and the results are highly reproducible. The techniques are easy to perform and do not require expensive reagents or equipment. The HA and IHA techniques therefore offer an alternative to other techniques for measuring ECR1 activity.

A dot-immunobinding assay for the demonstration of soluble Fc gamma receptors.

We have developed a sensitive dot-immunobinding assay to demonstrate and characterize the functional activity of soluble Fc gamma receptors (FcR). Samples containing soluble FcR were immobilized on a nitrocellulose membrane. Immune complexes of horseradish peroxidase and rabbit IgG antibodies to horseradish peroxidase (HRP) were allowed to react with nitrocellulose-bound FcR, and the immune complexes were visualized by HRP developer. The intensity of the grey dots reflected the amount of immune complex bound. Binding of immune complexes to placental extract containing soluble FcR was inhibited completely by IgG and Fc fragments, but not by F(ab')2 fragments, IgA and IgM. The method was used to characterize the subclass specificity of solubilized placental FcR. Human Fc fragments, and intact IgG1 and IgG3 proteins inhibited the binding whereas preparations of F(ab')2, IgG2 and IgG4 did not. In conclusion, the dot-immunobinding assay described is a rapid and simple method for the demonstration and characterization of functionally active soluble FcR.

Peripheral nerve CR1 limit complement-mediated haemolysis.

CR1 were purified from sciatic nerve extracts by monoclonal antibody affinity chromatography. The effect of CR1 on complement-mediated haemolysis was studied by adding purified CR1 to a mixture of human serum as source of complement and sheep erythrocytes sensitized with rabbit IgG. A dose-dependent inhibition of the haemolysis occurred. There was no effect on the haemolysis when phosphate-buffered saline, elution buffer or run-through fraction not containing CR1 was added. Addition of a polyclonal anti-CR1 antibody to purified CR1 suppressed the inhibiting activity, strongly indicating that peripheral nerve CR1 inhibit complement-mediated haemolysis. This may be of great importance in vivo since CR1 present on the Schwann cell membrane may prevent the formation of terminal lytic complexes.

Fc gamma receptors and HLA-DR antigens on thymus cells in myasthenia gravis.

Receptors for the Fc part of IgG (Fc gamma R) and HLA-DR antigens were detected in thymus tissue from patients with myasthenia gravis (MG) using monoclonal antibodies (B1D6 and OKIa1) in indirect immunofluorescence. The amount of Fc gamma R and HLA-DR antigens was increased on epithelial reticular cells and on interdigitating reticular cells in hyperplastic thymus as compared to normal thymus. In thymomas from MG patients the neoplastic epithelial cells expressed Fc gamma R, whereas only a few cells had HLA-DR antigens. Neither thymocytes nor B lymphocytes in the hyperplastic thymuses and in the thymomas were stained by B1D6.

Peripheral nerve CR1 express in situ cofactor activity for degradation of C3b.

Adsorption of sheep erythrocytes (E) sensitized with IgM antibodies (A) and C3b (EAC3b) to C3b/C4b receptors (CR1) in cryostat sections of human myelinated nerves was studied using the closed chamber technique. The adsorption was stable for at least 3 h at 37 degrees C. In the presence of purified factor I, the indicator cells detached from the sections after 40 min at 37 degrees C. Factor H was not required. The release was not due to loss of CR1 activity in the sections. The detached indicator cells were negative in the immune adherence test and were agglutinated by antibody to C3d, but not by antibody to C3c. Western blot of the detached indicator cells revealed the presence of C3d and C3c was found in the chamber fluid. Accordingly, detachment of the indicator cells was due to degradation of C3b to C3d with the release of C3c into the chamber fluid. Protease inhibitors did not prevent the detachment of the indicator cells. EAC3b incubated with sections of myelinated nerves pre-incubated with anti-CR1 antibody or with sections of unmyelinated nerves which contain functionally inactive CR1 were not degraded. The results therefore indicate that CR1 in situ in myelinated nerves can provide the necessary cofactor activity for factor I-mediated degradation of C3b to C3d and C3c.

Complement receptors CR1 on human peripheral nerve fibres.

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 significant differences in the binding of E-bearing C3b or C4b to myelinated peripheral nerves from 50 individuals, indicating that CR1 activity is not distributed phenotypically.

Myasthenia gravis. Antibodies to skeletal muscle cell surface antigens.

Sera from 28 of 137 patients with myasthenia gravis (MG) (i.e. 20%) contained antibodies which stained the surface of skeletal muscle cells in an indirect immunofluorescence test. Forty of the 137 sera (i.e. 30%) contained cross-striational antibodies. Absorption experiments showed that the antibodies staining the muscle cell surface were different from those staining the cross-striational bands. Twenty of the sera (i.e. 15%) contained antibodies which agglutinated sheep erythrocytes (SE) coated with a citric acid extract of skeletal muscle (CAE). These antibodies were closely associated with the presence of a thymoma. There was a positive correlation between the antibodies agglutinating CAE-coated SE and those staining the muscle cell surface. Absorption experiments indicated that the antibodies to CAE were directed against muscular antigens located in or near the sarcolemma.

Localization of Fc gamma receptors and complement receptors CR1 on human peripheral nerve fibres by immunoelectron microscopy.

The localization of receptors for the Fc part of IgG (Fc gamma R) and for the complement C3b/C4b components (CR1) on human peripheral nerve fibres was investigated by indirect immunoperoxidase staining of frozen nerve sections with monoclonal antibodies. Transmission electron microscopy revealed that Fc gamma R and CR1 are localized to the entire surface membrane and inner membrane (axolemma) of the Schwann cell. Myelin and axons were not stained. The presence of Fc gamma R and CR1 in human Schwann cells adds further evidence for the immunocompetence of these cells.

Isolation and characterization of complement receptors CR1 from human peripheral nerves.

Extracts from myelinated and unmyelinated nerves, prepared using Nonidet P-40, contained receptors for C3b/C4b (CR1). Extracts from myelinated nerves inhibited EAC3b rosette formation with peripheral blood leucocytes and agglutinated EAC3b, whereas extract from unmyelinated nerves did not. Rosette formation with EAC3bi or EAC3d was not affected. CR1 in extracts from myelinated nerves also expressed decay-accelerating activity of the alternative pathway C3 convertase and cofactor activity in factor I-mediated cleavage of C3b, whereas CR1 in extract from unmyelinated nerves did not. Monoclonal anti-CR1 antibodies, but not monoclonal anti-CR2 (C3d receptors) or anti-CR3 (C3bi receptors) antibodies inhibited the functional activities. Accordingly, CR1 are the only C3 receptor present in the extracts and only CR1 in myelinated nerve extracts are functionally active. CR1 in both myelinated and unmyelinated nerve extracts had a molecular weight of approximately 190 kDa. The electrophoretic mobility did not change after reduction and the 190 kDa band was stained by concanavalin A, indicating that the CR1 are single-chained glycoproteins. Binding to lentil lectin-Sepharose 4B further sustained the glycoprotein nature of the CR1. Periodic acid abolished functional activities of CR1, whereas trypsin and heat did not, indicating the functional significance of the carbohydrate moiety. That CR1 are functionally active in myelinated nerves, but not in unmyelinated nerves, may therefore be due to differences in the carbohydrate moiety. The cofactor and decay-accelerating activities of CR1 may be of significance in the pathogenesis of demyelinating polyneuropathies by limiting complement activation.

Antibodies to peripheral nerve tissue in sera from patients with acute Guillain-Barré syndrome demonstrated by a mixed haemagglutination technique.

The mixed haemagglutination technique (MA) was used to demonstrate antibodies to peripheral nerve tissue (PNS) in sera from patients with acute Guillain-Barré syndrome (GBS). Within the first 2 weeks of the disease IgG antibodies were detected in sera from 15 of 42 patients (36%). Antibodies were detected in 60% of the patients with severe GBS, and in 30% of those with a mild GBS. The highest titres were found in the sera from the most severely affected patients and could be detected after 48 days of the illness in one of the sera. Sera from healthy blood donors and from patients with various other diseases did not contain antibodies to PNS. The MA technique is reproducible and is more sensitive than the indirect immunofluorescence test and the antiglobulin consumption test.

Rabbit antiserum to a citric acid extract of human skeletal muscle staining thymomas from myasthenia gravis patients.

Rabbit antiserum to a citric acid extract of human skeletal muscle (CA) stained both the cell membrane and the cross-striational bands of skeletal muscle cells. The rabbit antiserum also stained the cell membrane of epithelial thymoma cells from myasthenia gravis (MG) patients. Normal and hyperplastic thymus tissue were not stained, apart from scattered myoid thymic cells. Absorption of the antiserum with CA abolished staining of the thymoma, indicating that human skeletal muscle and epithelial thymoma cells possess common antigens.

Soluble complement receptor type 1 in serum and cerebrospinal fluid of patients with Guillain-Barré syndrome and multiple sclerosis.

Activation of complement is critically involved in inflammatory reactions in both Guillain-Barré syndrome (GBS) and multiple sclerosis (MS). Soluble human complement receptor 1 (sCR1) blocks complement activation by both classical and alternative pathways. We studied serum and cerebrospinal fluid (CSF) concentrations of sCR1 in 23 patients with GBS, 27 patients with MS and 30 controls. No significant differences were found between patients and controls. Transient liver affection probably caused high serum sCR1 levels in two patients with GBS. The serum and CSF sCR1 levels were not correlated to the disease activity of GBS and MS, nor to the relapsing-remitting or chronic-progressive forms of MS. In GBS the CSF sCR1 levels correlated with the CSF total protein concentrations (r = 0.9, P < 0.01), suggesting that sCR1 leaks from serum into CSF via a damaged blood-nerve barrier. The serum sCR1 levels in GBS were slightly higher than in MS (P < 0.05). Whether this reflects changes in the release or consumption of sCR1 in these patients is at present unknown.

Skeletal muscle antibodies in patients with a thymic tumour but without myasthenia gravis.

Sera from 9 patients with a primary thymic tumour but without myasthenia gravis (MG) at the time of thymectomy were examined for skeletal muscle antibodies. Antibodies to a citric acid extract (CA), associated with the presence of a thymic lymphoepithelioma in MG patients, were detected in 3 sera. AChR antibodies were detected in 5 sera, the concentration markedly elevated in 1 of them. Sarcolemmal and cross-striational antibodies were detected in 2 and 3 sera, respectively. Four of the 5 patients with thymic tumours other than a lymphoepithelioma had AChR antibodies, and 3 of them also had CA antibodies.

Fc receptor for IgG (FcR) on rat microglia.

Receptor for IgG (FcR) was demonstrated on rat microglia in vivo and in vitro by immunohistochemical staining with immune complexes of horseradish peroxidase (HRP) and rabbit IgG anti-HRP. Astrocytes, oligodendrocytes and neurons did not express FcR. Microglia in culture also showed FcR-mediated agglutination and phagocytosis of IgG-sensitized erythrocytes. A radiolabelled cDNA probe for rat FcRIII hybridized with a 1.4-kb RNA band in Northern blots prepared from total RNA from rat brain. FcRIII mRNA-positive cells in rat brain, presumably microglia, were demonstrated by in situ hybridization. FcR participates in the initiation of cytotoxic responses and of phagocytosis by microglia and is therefore likely to be important in mediating immune reactions in the brain.