IVIG blocks complement deposition mediated by anti-GM1 antibodies in multifocal motor neuropathy.

BACKGROUND: The pathogenesis of multifocal motor neuropathy (MMN) has yet to be established. MMN patients often carry anti-GM1 IgM antibodies, suggesting an autoimmune process involving complement. Intravenous immunoglobulin (IVIG) is the first line treatment, but its action mechanism is unknown. OBJECTIVE: To test whether anti-GM1 IgM antibodies in MMN sera activate complement, inducing and propagating the disease and whether IVIG inhibits complement activation, resulting in clinical improvement. METHODS: Sera with anti-GM1 IgM but not IgG or IgA reactivity were obtained from 13 patients with MMN. We tested whether their anti-GM1 IgM antibodies produced complement component deposits on GM1-coated microtiter plates and whether IVIG blocks such deposition. RESULTS: C1q, C4b, C3b and C5b-9 were deposited on GM1-coated wells. Their depositions were highly correlated with anti-GM1 IgM antibody titre. IVIG reduced the deposition of these complement components dose-dependently. CONCLUSIONS: Anti-GM1 IgM antibodies bound to GM1 and activated complement in vitro. The results together with earlier data from our group suggest that IgM-induced, complement-mediated injury occurs at the nodes of Ranvier in peripheral motor nerves and generates conduction block and muscle weakness. In vitro IVIG inhibited this type of complement activation, suggesting that in vivo, the resulting reduction in membrane attack complex-mediated damage leads to improved muscle strength.

Recurrent meningitis in a patient with congenital deficiency of the C9 component of complement. First case of C9 deficiency in Europe.

We describe the first cases, to our knowledge, of C9 deficiency in Europe that were detected in a Swiss family, of which two members--one with a complete deficiency and the other with approximately half-normal C9 levels--experienced bacterial meningitis. The index patient, a 56-year-old white man with a history of purulent meningitis at the age of 23 years, presented with an acute meningococcal meningitis. No impairment of cellular immunity or immunoglobulin deficiency could be found. Complement assays showed a complete deficiency of the C9 component, while the other individual component levels were normal and the hemolytic activity (measured using the CH50 assay) was only slightly reduced. A family study revealed complete C9 deficiency in the patient's healthy brother and half-normal C9 concentrations in his sister, his son (who also had experienced an episode of bacterial meningitis), and his niece, consistent with an inherited C9 deficiency. This first case of recurrent meningitis in a white patient with complete C9 deficiency suggests that this complement defect may also be a risk factor for bacterial, especially neisserial, infections.

Immunoglobulin-mediated shifting of immune complexes to increased complement-dependent solubility and immunoadherence.

The effect of a monomeric polyclonal polyspecific IgG preparation (mIgG) for i.v. use on the capacity of fresh normal human serum (NHS) to inhibit precipitation of immune complexes of tracer-labeled bovine serum albumin (BSA) and anti-BSA (aBSA) rabbit antibody was investigated. Relative to heat-inactivated serum which showed no capacity to inhibit immune complex precipitation (CIICP), fresh NHS at a final dilution of 1:3 or 1:2 showed a 12 and 46% CIICP, respectively, with a BSA:aBSA ratio at equivalence. Addition of incremental amounts of mIgG dose-dependently enhanced CIICP of NHS to reach 63 and 90%, respectively, at a concn of 13.0 mg/ml mIgG. Human serum albumin instead of mIgG had no influence on CIICP of NHS indicating that the phenomenon was not dependent on non-specific protein-protein interactions. Although minimal amounts of BSA cross-reactivity could be demonstrated in mIgG, the extent of this activity was too small to explain the CIICP-supporting effect of mIgG as determined by comparing the effect of mIgG and aBSA serum on CIICP of fresh serum. Furthermore, absorption of mIgG on BSA-Sepharose did not lead to impairment of its CIICP-supporting effect. A direct binding of tritium-labeled mIgG (3H-mIgG) to either insoluble BSA:aBSA complexes or latex-bound IgG (IgG-latex) was found. Incubation of 2.7 micrograms 3H-mIgG with 90 micrograms IgG-latex resulted in a specific binding of 5.2% of the labeled compound. Such binding could be inhibited by unlabeled mIgG. Binding of mIgG was mediated through the Fab as well as the Fc part of the molecules: the 3H-Fc as well as the 3H-Fab fragment of mIgG bound to IgG-latex. The extent of binding of Fc was more than twice that of Fab and amounted to 70-90% of the binding found with intact mIgG. In an immune adherence hemagglutination system that depends on the interaction of complement-reacted immune complexes with C3b receptor-bearing erythrocytes, evidence was obtained that the mIgG preparation facilitated fixation of C3b to performed BSA:aBSA complexes. Addition of 1.45 mg/ml mIgG reduced the quantity of antigen-complexed C3b-bearing aBSA antibodies required to show a given agglutination by a factor of 3-4. We conclude that the facilitating effect of high doses of mIgG on complement-dependent CIICP of NHS and on immune adherence hemagglutination is the amplifying effect of complement after an initial interaction of antigen-nonspecific polyclonal polyspecific IgG with antigen-reacted antibody.

Solubilization of immune precipitates by complement in the absence of properdin or factor D.

Various experiments have demonstrated that immune precipitates (IPs) are not solubilized by complement in the absence of alternative pathway function. To determine whether the characteristics of the IPs were responsible for these observations, we studied the solubilization (Sol) of IPs formed by bovine serum albumin (BSA)-rabbit antiBSA and tetanus toxoid (TT)-human antiTT. Sera deficient in properdin solubilized a fraction of BSA-antiBSA precipitates, although only when the IPs were formed in antibody excess. The same sera solubilized TT-antiTT precipitates with some delay but almost as efficiently as normal serum. Factor D-depleted serum solubilized a fraction of TT-antiTT precipitates too, indicating that Sol may proceed through activation of the classical pathway only. Thus, the requirements for complement-mediated Sol depend on the characteristics of the IPs and do not necessarily include alternative pathway function.

Human serum induced opsonization of immunoglobulin G-coated polystyrene microspheres with complement components C3 and C4 as measured by flow cytometry.

Human IgG-coated polystyrene microspheres (IgG-ms) were incubated with human serum followed by biotinylated monoclonal anti-C3d or anti-C4d antibody, and phycoerythrin-streptavidin. The intensity of fluorescence was measured by flow cytometry and corresponds to the amount of deposited C3 and C4. Binding of C3 and C4 was dependent on the activation of the classical pathway of complement and on the amount of IgG adsorbed to the particles. No deposition was observed on control particles coated with bovine serum albumin or ovalbumin. Incubation of constant amounts of IgG-ms with increasing amounts of normal human serum (NHS) resulted in a dose-dependent increase in C3 deposition. The same result was found for C4 deposition at moderate NHS dilutions, but less C4 was detectable using a higher input of NHS. Half-maximum C3 and C4 deposition was observed at a mean serum dilution of 1/114 and 1/520, respectively (n = 26). No correlation was found between C4 or C3 deposition and either total C4 and C3 serum concentrations as measured by nephelometry or complement-mediated lysis of antibody-coated sheep red blood cells. Reduced or absent C4 or C3 deposition was found in the sera of patients with low amounts or deficiencies of components involved early in classical complement pathway activation whereas essentially normal C4 or C3 deposition was obtained with the sera of patients with deficiencies in components of the membrane attack complex. With this simple and specific functional assay using stable reagents an altered function of early components of the classical pathway of complement may be quickly and reliably detected in routine diagnostic laboratories. Moreover, such opsonized and well characterized particles may be useful in assays of phagocytic cell function.

A C3 convertase assay for nephritic factor functional activity.

C3 nephritic factor (C3NeF) is an autoantibody against the C3 convertase which stabilizes this otherwise inherently labile neoenzyme and induces a continuous activation of the alternative pathway with C3 depletion. NeF is found in patients with membranoproliferative glomerulonephritis and/or partial lipodystrpohy. NeF activity is usually detected in plasma by hemolytic tests. In order to obtain reproducible data for the functional activity of purified C3NeF IgG a solid phase assay was developed. C3 convertase was generated on immobilized C3b by incubation with factors B and D in the presence of Ni(2+). Convertase sites were left to decay in the presence of normal IgG or NeF IgG. Residual convertase activity was measured by adding 125I-C3 and capturing nascent 125I-C3b on the plate surface via covalently coupled NH2-Glu-Tyr dipeptide. In the presence of factor H during C3 convertase decay, a dose dependent stabilizing activity was shown for NeF IgG including NeF IgG purified from urine. A second format of the assay was developed in which C3 convertase was assembled on C3b(2)-IgG complexes in the presence of Mg(2+). Since these complexes are more efficient as convertase precursors the signal was five-fold higher than with C3b. Convertase decay, on the other hand, was not influenced by the nature of the precursor and in both systems the stabilizing activity of NeF IgG was similar.

Hypocomplementemic urticarial vasculitis or systemic lupus erythematosus?

The 2 patients presented here showed the typical signs of hypocomplementemic urticarial vasculitis syndrome (HUVS). During follow-up, there was an inverse correlation between anti-C1q autoantibody titer and C1q antigen concentration in serum in both patients over a period of 2 years. The first patient had nephritis characterized by immune deposits in glomeruli and around the tubules. The histological findings, C1q deposits, and presence of tubuloreticular inclusions in capillary endothelial cells suggested a disease process identical to systemic lupus erythematosus (SLE). The second patient, after a lag phase of 2 years, fulfilled a fourth American College of Rheumatology criteria for SLE when she developed anti-double-stranded DNA antibodies. HUVS and SLE overlap, and the criteria for identifying HUVS as an entity distinct from SLE are lacking.

Glomerulonephritis in a patient with complement factor I deficiency.

Complement factor I deficiency is known to be associated with recurrent pyogenic infections. The patient described here had recurrent attacks of otitis, sinusitis, and bronchopneumonia since childhood. At the age of 24 years, he had an acute episode of systemic vasculitis with purpura, but no nephritis. A factor I deficiency was diagnosed when he was 36 years old. Because of the uncontrolled activation of the alternative pathway of complement, several other components were depleted, in particular C3, which explained the predisposition for pyogenic infections. A progressive loss of renal function accompanied by proteinuria and hematuria started after the age of 40 years. Renal biopsy showed a focal segmental glomerulonephritis (GN) with glomerular deposits of immunoglobulins and complement C3 and C4 fragments. The glomerular podocytes showed an almost complete loss of complement receptor 1 (CR1; CD35). The expression of CR1 was very low on erythrocytes, as well. Thus, CR1, the most efficient cell-bound cofactor for the inactivation of C4b/C3b by factor I, appears to be consumed when factor I is missing. Although this is the first report of factor I deficiency associated with GN, it is unlikely that the development of the nephritis was fortuitous because GN has been found in many other diseases characterized by uncontrolled activation of the alternative pathway.

Genetic detection of the silent allele (*Q0) in hereditary deficiencies of the human complement C6, C7, and C9 components.

DNA polymorphisms (RFLPs) of the human complement component C6, C7, and C9 genes were studied in three C7-deficient (C7D) families, one C6-deficient (C6D) family, and one C9-deficient (C9D) family. The 3 loci are closely linked on human chromosome 5. The haplotypes carrying the "silent" allele (C7*Q0, C6*Q0, and C9*Q0) were defined in each family, allowing for the detection of carriers among asymptomatic relatives. This paper describes familial studies on a type of hereditary trait, characterized by recurrent Neisseria infections in individuals homozygous for "silent" alleles at the C6, C7, or C9 loci.

Hereditary complement factor I deficiency.

We describe four cases (from three families) of hereditary factor I deficiency, bringing the total number of cases now reported to 23. In one family there are two affected siblings: one has suffered recurrent pyogenic infections; the other is asymptomatic. In the second family, the patient had recurrent pyogenic infections and a self-limiting vasculitic illness; in the third family, the patient suffered recurrent pyogenic and neisserial infections. All four patients had markedly reduced concentrations of C3 in the serum (family 1 propositus: 28%; family 1 asymptomatic sibling: 15%; family 2: 31%; and family 3: 31% normal human serum) which was in the form of C3b. Low IgG2 levels may occur in primary C3 deficiency, and a reduction in IgG2 concentration to 1.14 g/l (normal: 1.30-5.90 g/l) was found in the patient from family 2. Using radioligand binding assays, we demonstrated increased binding of C3b to erythrocytes in a patient with factor I deficiency. This C3b could not be cleaved by autologous serum but could be cleaved by normal serum or purified factor I. We review and compare the published cases of C3, factor H and factor I deficiency.

Complement profiles in human skin lymph during the course of irritant contact dermatitis.

Using microsurgery a superficial peripheral lymph vessel draining the skin of the upper and medial part of the foot was cannulated on the lower leg of two healthy human volunteers. An irritant contact dermatitis was induced 2 days later by the application of 10% sodium lauryl sulphate to the drained skin area. After a further 3 days the spontaneously regressing skin reaction was treated with clobetasol propionate. Lymph was continuously collected in two aliquots per day for 7 days. The levels of total protein, of albumin and globulins, and of complement components of the classical, the alternative and the lytic pathway as well as the C4A and C4B gene products and the regulatory proteins FB, C1INH, C4BP, FH and FI were determined by ELISA and radial immunodiffusion techniques. Postoperatively, the levels of complement proteins and globulins in the lymph were 5-10 times lower than those in normal human serum, but increased during the course of the skin reaction, while the irritant contact dermatitis did not induce a change in their plasma concentration. In comparison to the baseline, the mean values for C1q, C1r, C2, C5, C6, C7, C8, C9, FB, C1INH, C4BP, FH and FI exhibited a 3-5-fold increase, C3, total C4, albumin and the alpha 1-globulin fraction a 6-9-fold increase, and C1s, C4A, C4B, FB and alpha 2-, beta- and gamma-globulins a 10-20-fold increase.(ABSTRACT TRUNCATED AT 250 WORDS)

High doses of antigen-nonspecific IgG do not inhibit pemphigus acantholysis in skin organ cultures.

A patient suffering from severe pemphigus vulgaris was treated using large-volume plasma exchange in combination with an immunosuppressive regimen. As some recent reports have shown evidence that polyclonal, polyspecific human IgG in high doses through the i.v. route (IGIV) protect target platelets in idiopathic thrombocytopenic purpura from attack by antiplatelet autoantibodies and/or immune complexes, we also administered IGIV to this pemphigus-vulgaris patient. In order to test the hypothesis that IGIV might protect in vitro-cultured human skin from acantholysis induced by pemphigus antibodies, studies with skin organ cultures were carried out using plasma from another pemphigus-vulgaris patient who had undergone plasma exchange. The preincubation of either the skin explants or the pemphigus plasma with various concentrations of IGIV (ranging from 0.15 to 15 mg/ml in the culture medium) did not prevent acantholysis induced by the pemphigus plasma nor did it inhibit the binding of the specific antibodies visualized by direct immunofluorescence. Thus, the assumption that IGIV may coat the pemphigus antigens on epidermal cells making them inaccessible to pathogenic autoantibodies was not substantiated by our tests in vitro; likewise, the hypothesis of functionally blocking autoantibody activity by means of anti-idiotype effects of IGIV cannot be supported.

Humoral immune response to pneumococcal antigen 23-F in an asplenic patient with recurrent fulminant pneumococcaemia.

Host defence mechanisms were analysed in a patient with three episodes of fulminant pneumococcaemia and one episode of bacteraemic epiglottitis with Haemophilus influenzae type b. The first episode took place 11 years after splenectomy for blunt abdominal trauma. Investigations revealed several host defence mechanisms to be impaired. In addition to the patient's asplenia, an inherited C2-deficiency was noted. Assessment of IgG subclasses repeatedly revealed markedly low IgG4 concentrations. These were not due to an increased turnover of IgG4, as could be shown following infusion of intravenous IgG. In addition, IgG2 concentrations were low in the patient who lacked G2M(23). Opsonic mediating antibodies against type 23-F pneumococci were in the range of those of non-immune volunteers 6 months after vaccination with a 23-valent pneumococcal vaccine. These antibodies did not increase after a septic episode with 23-F capsular-type pneumococci. Neutrophil function was apparently normal.

Angioedema. A review on the acquired, allergic or non-allergic, and the hereditary forms.

This review intends to review the various clinical and biochemical backgrounds of angioedema: i.e. angioedema in association with allergic or pseudoallergic reactions and the cells involved as well as angioedema on the basis of functional deficiencies of regulatory proteins such as C1-esterase-inhibitor, plasma carboxypeptidase B, and the angiotensin-converting enzyme. Angioedema in suggested association with sexual hormone balance shifts is another topic discussed. The manuscript further summarizes some possible therapies and laboratory practices for diagnostic purposes of the various forms of angioedema.

IVIG--mechanisms of action.

Intravenous immunoglobulin (IVIG) preparations are fractionated from a plasma pool of several thousand donors. IVIG contain immune antibodies and physiologic autoantibodies. Immune antibodies reflect the immunologic experience of the donor population. This fraction of IVIG preparations is useful for replacement therapy and passive immunisation. Natural autoantibodies are able to react with the immune system of the recipient of IVIG and are suggested to help to correct immune deregulation. Immunomodulatory and anti-inflammatory properties are based on multiple mechanisms of action which are described. These mechanisms are effective concomitantly and synergistically at every occasion of use of IVIG in inflammatory and autoimmune disorders.

Quantification of complement receptor 1 on erythrocytes: follow-up of HIV-1-infected patients with AIDS-related complex/Walter-Reed 5 under treatment with intravenous immunoglobulin. The ARC-IVIG Study Group.

A sensitive flow-cytometric method was established to quantify the number of complement receptor 1 (CR1, C3b/C4b receptor, CD35) on the surface of purified erythrocytes of 12 patients infected by HIV-1 and showing two clinical AIDS-related complex/Walter-Reed 5 criteria. Erythrocytes were incubated with biotinylated monoclonal anti-CR1 antibody followed by phycoerythrin-streptavidin before analysis on a flow cytometer. As few as 50 binding sites/cell could be detected, making this method as sensitive as a radioimmunoassay with 125I anti-CR1. Seven of the patients studied received an immunoglobulin preparation suitable for intravenous use during the 6 months of the study, 5 got an equal amount of placebo preparation consisting of human serum albumin. For a year, erythrocytes were collected and purified every 3 months, frozen and stored at -70 degrees C until the end of the study, when the number of CR1 was determined. No difference between the two groups of patients was found. In 8 patients, small fluctuations of the amount of CR1/erythrocyte were seen during the period of observation, whereas in 4 of the patients a drop of the number of CR1 was observed towards the end of the study. No correlation was found between CR1 numbers on erythrocytes and circulating immune complexes, CH50, C3 and C4 concentrations or CD4-positive lymphocytes.

Recurrent angioedema associated with hypogonadism or anti-androgen therapy.

Two male patients with hypogonadism and four female patients who received an anti-androgen as contraceptive (cyproteronacetate) and who had recurrent angioedema are described. In one male patient, augmentation of the plasma androgen level resulted in disappearance of symptoms. In the four female patients, recurrent angioedema and urticaria developed after initiation of the anti-androgen treatment. Cessation of cyproteronacetate and a change to another contraceptive resulted in complete resolution of the previously frequent angioedematous attacks. The women are still symptom free after more than 60 patient's months. These cases suggest that an androgen deficit due to either hypogonadism or to anti-androgen treatment may be another cause of angioedema. One of the two male patients was untreated and presented with 40% normal value of C1-INH. Androgen therapy normalized C1-INH concentration in this male patient. Functional C1-INH in the same patient, studied before and after the beginning of androgen therapy, clearly increased when assessed by inhibition of amidolytic activity of C1-esterase. The other male patient with hypogonadism had already been under androgen treatment for 4 years and had C1-INH levels in the normal range. In the female patients, complement profiles were normal before and after cessation of anti-androgen contraception; however, the C1-INH plasma levels were higher after cessation of anti-androgen anticonception. These results indicate an effect of androgen deficit on the level of C1-INH in circulating plasma but do not prove a role of C1-INH in angioedema associated with diminished androgen plasma levels.