Neutrophil lactoferrin release induced by IgA immune complexes differed from that induced by cross-linking of fcalpha receptors (FcalphaR) with a monoclonal antibody, MIP8a.

The human IgA Fc receptor (FcalphaR, CD89) plays an important role in host defence against invading pathogens. To study the properties of the receptor, 12 MoAbs, namely, MIP7c, MIP8a, MIP9a, MIP10c, MIP11c, MIP14b, MIP15b, MIP38c, MIP59c, MIP65c, MIP68b and MIP71a, were generated. The inhibitory effects of the antibodies on FcalphaR functions were tested. Three of the antibodies, MIP7c, MIP8a and MIP59c, were able to block up to 90% of soluble FcalphaR binding to IgA-coated beads and 70-80% of neutrophil phagocytosis of IgA immune complexes (IC). MIP8a could also inhibit IgA IC-induced neutrophil lactoferrin release, while cross-linking of FcalphaR with MIP8a and anti-mouse IgG could elicit neutrophil lactoferrin release. However, IgA IC-induced lactoferrin release required both extracellular calcium and magnesium, whereas MIP8a-induced release did not require extracellular magnesium and only partially required extracellular calcium. In addition, the time course of IgA IC-induced lactoferrin release was slow. Lactoferrin was not detectable if the incubation time was less than 0.5 h. In contrast, MIP8a-induced lactoferrin release was fast. Lactoferrin could be detected within 5 min of incubation. Therefore, neutrophil lactoferrin release induced by IgA IC differed from that induced by cross-linking of FcalphaR with MIP8a.

Difficulties in the ascertainment of C9 deficiency: lessons to be drawn from a compound heterozygote C9-deficient subject.

A group of patients with long-surviving mismatched kidney allografts were investigated for complement function using haemolytic assays in agarose gels. One patient was found to have no alternative pathway activity but a low normal classical pathway. Surprisingly, investigation revealed that the patient's complement was normal for all components except C9, which was functionally absent. The patient was shown to be heterozygous for DNA markers in the C6, C7 and C9 region of chromosome 5 and therefore appears to be a compound heterozygote for two uncharacterized C9 deficiency genes. Serological analysis by ELISA revealed that he has trace concentrations of a non-functional C9 molecule. Western blot analysis was not sufficiently sensitive to permit detection of this molecule. We hypothesize that the patient is heterozygous for a complete deficiency of C9 and for a gene directing hyposynthesis of a defective C9. We also suggest that C9 deficiency may be more common among Caucasians than has been reported.

Effects of decomplementation with cobra venom factor on experimental vasculitis.

Mercuric chloride (HgCl2) induces autoimmunity in susceptible rat strains, with hyper-IgE, appearance of a number of autoantibodies, and widespread tissue injury, including necrotizing vasculitis in the gut. In the early phase of tissue injury there is granulocyte infiltration; later there is immunoglobulin deposition along basement membranes in vessels. We have analysed the role of complement in this model using cobra venom factor (CVF), which causes decomplementation lasting around 5 days. The characteristic time course when HgCl2 is given over 10 days is that tissue injury and autoantibody levels reach a peak at around day 15 (start of HgCl2 = day 0). We therefore gave CVF either early (day 0), intermediate (day 5) or late (day 10); a fourth group (controls) received HgCl2 but no CVF. At each time point, CVF caused complete decomplementation which lasted for at least 5 days. Serum IgE and autoantibody levels were similar in all four experimental groups. Tissue injury in the 'early' CVF group and in the 'late' CVF group was not significantly different from controls, but in the intermediate group tissue injury was significantly more severe than in controls. These data indicate that the complement system does not play a major role in the induction of autoantibodies by HgCl2, nor in the effector phase of tissue injury. We speculate that the exacerbation of tissue injury by CVF in the group given this agent at an intermediate stage of the model is explained by the presence of products of C3 activation which have proinflammatory effects during the phase of active granulocyte-mediated tissue injury.

Measuring the number of lamellar body particles in amniotic fluid.

We describe a method for determining the number and size distribution of lamellar bodies and compare the results prospectively with other tests for fetal lung maturity: lecithin-sphingomyelin ratio (L/S), phosphatidylglycerol, and fluorescence polarization. The technique uses an electronic particle counter calibrated for a size range of 1.7-7.3 fL. The number of lamellar bodies in amniotic fluid samples varied from 3800-166,000 particles per microliter and correlated strongly with L/S ratio (r = 0.75; N = 144) and fluorescence polarization (r = -0.78; N = 165). Amniotic fluid samples stored for up to 10 days at 4C had stable lamellar body counts (within +/- 11%). Longer storage tended to decrease the counts. Addition of more than 1% (v/v) whole blood significantly decreased the lamellar body counts. This technique shows promise for the rapid assessment of fetal lung maturity.

Bovine serum conglutinin is a lectin which binds non-reducing terminal N-acetylglucosamine, mannose and fucose residues.

Carbohydrate recognition by bovine serum conglutinin has been investigated by inhibition and direct binding assays using glycoproteins and polysaccharides from Saccharomyces cerevisiae (baker's yeast), and neoglycolipids derived from N-acetylglucosamine oligomers, mannobiose and human milk oligosaccharides. The results clearly show that conglutinin is a lectin which binds terminal N-acetylglucosamine, mannose and fucose residues as found in chitobiose (GlcNAc beta 1-4GlcNAc), mannobiose (Man alpha 1-3Man) and lacto-N-fucopentaose II [Fuc alpha 1-4(Gal beta 1-3)GlcNAc beta 1-3Gal beta 1-4Glc] respectively.

Use of monoclonal anti-C3 antibodies to characterise the fragments of C3 that are found on erythrocytes.

Using monoclonal antibodies to C3 it has been shown that the red blood cells of patients with cold haemagglutinin disease carry on their cells C3d,g (alpha-2D-globulin) rather than C3d. C3d,g seems to be the final product of in vivo C3 activation in fluid phase and on red cells. The cleavage of C3dg to C3d and C3g does not appear to occur in vivo either in the fluid phase or on red cell bound C3bi. In vitro C3-coated red cells prepared by antibody or low ionic strength techniques produce cells with C3d and C3bi as the predominant C3 fragment, whereas the Fruitstone technique in which coating occurs by the alternative pathway has principally C3b. The activity of C3 cleaving enzymes in whole serum is strongly influenced by the ionic conditions of the serum.

Breakdown of C3 after complement activation. Identification of a new fragment C3g, using monoclonal antibodies.

The physiological breakdown of C3 has been studied using monoclonal anti-C3 antibodies, and it has been found that the later stages of this process--the breakdown of C3bi--is more complex than had previously been recognized. C3bi is the reaction product produced from C3b by the action of factor I which, in the presence of factor H, produces a double cleavage in the alpha chain of C3b. It is here reported that, both on cells and in the fluid phase, the breakdown of C3bi in serum gives rise to two products: C3c and the product previously described as alpha 2D, which we now propose to designate C3d,g. Alpha 2D differs from C3d in that it contains an additional fragment of approximately 8,000 mol wt that carries the antigenic determinant for the clone 9 monoclonal anti-C3 antibody. C3g cannot be precipitated by anti-C3 antisera and therefore behaves as a uni- or bideterminant antigen. The cleavage of C3d,g to C3d and C3g does not occur in sterile serum. It is also still uncertain what enzyme cleaves C3bi to C3c and C3d,g in plasma. Plasmin can do so in vitro, but plasminogen-depleted serum can still produce the cleavage. The antigenic determinant recognized by clone 9 in C3 is not exposed in C3 or C3b, but appears as a neoantigen in C3bi (and in C3d,g). Anti-C3g therefore is a potentially useful ligand for detecting complement-activation products. C3g represents a new, highly anionic C3 fragment and seems not to be identical with the C3e fragment described by others.

The isolation of the antibody moieties of immune complexes from serum by the pepsin digestion of conglutinin-anti-conglutinin complexes.

A technique is described which allows the antibodies of circulating immune complexes to be isolated as their F(ab')2 fragments. The method is based on the precipitation of the complexes by the sequential addition of conglutinin and anti-conglutinin, and the subsequent digestion of these precipitates by pepsin. Using this technique it has been possible to show antibodies to Epstein-Barr (EB) virus antigens in the immune complexes of patients with Burkitt's lymphoma and to microbial antigens in two patients with nephritis. By substituting DNAase for pepsin it has also been possible to show antibodies to DNA-containing nuclear antigens in the serum of patients with systemic lupus erythematosus.

Three rat monoclonal antibodies to human C3.

Three monoclonal antibodies to human C3 have been obtained from a fusion of the rat myeloma line Y3 Ag 1.2.3. with spleen cells from rats immunized against C3. One, from clone 4, reacts with an antigenic determinant in C3c showing the expected reactivity of the 'C' antigen of C3. The specificity of the other two monoclonal antibodies correspond less clearly with known C3 antigens. By agglutination analysis of complement coated cells the determinant reacting with clone 3 is present in C3d while that for clone 9 appears as a neoantigen on C3bi. In both cases the co-precipitation results are anomalous and more direct studies are needed to define the exact specificity. The possibility that internal sequence duplications in C3 may explain some anomalies is discussed. None of the monoclonal antibodies significantly inhibit C3 functions. The monoclonal antibodies have been found to have unusual properties in co-precipitation assays being able to diffuse through a precipitation line with which they react to react with a further line. One antibody is also able to react strongly with the anodal half of what appears as a single line with a polyclonal antiserum.