In an effort to identify the protein structure on Candida albicans, pseudohyphal forms which had been shown earlier to bind human iC3b, a protein of about 42 kDa (p42), were obtained from lysates of pseudohyphal forms by absorption with C3(H20)-Sepharose. An antiserum raised in rabbits against this protein effectively inhibited adherence of sheep erythrocytes carrying iC3b (EAC3bi) to pseudohyphal forms. p42 cross-reacted with OKM-1, a monoclonal antibody directed against the human complement receptor type 3 (CR3, CD11b). This protein, p42, was designated p42-CR3. The antiserum against p42-CR3 was used for further purification of lysates by affinity chromatography. Three proteins of 66, 55, and 42 kDa were isolated. All were recognized by OKM-1 in immunoblots (p66-, p55-, and p42-CR3). The different proteins were separated and treated with neuraminidase and endoglycosidase F. Almost complete deglycosylation of the p66-CR3 protein was obtained after treatment with neuraminidase, indicating a high degree of glycosylation. Neuraminidase also had an effect on p55-CR3, but not on p42-CR3. Endoglycosidase F did not alter any of the three proteins. In ligand blots, p42-CR3 bound C3(H20), C3b, and iC3b but not C3d; p55-CR3 clearly reacted with C3(H20) and weakly reacted with C3b and iC3b. p66-CR3 never showed reactivity. It is suggested that p55 and p66 represent glycosylated forms of p42-CR3. Although C. albicans CR3 and human CR3 cross-react and bind identical ligands, the two receptors differ in structure. Candida albicans pseudohyphal forms, in contrast to yeast forms, bind sheep erythrocytes carrying iC3b or C3d (EAC3bi, EAC3d). This observation, initially described by Heidenreich and Dierich (10), was confirmed later by several other investigators (3, 4, 6). Eigentler et al. (5) have shown that the expression of the iC3b receptor on C. albicans is dependent on the growth temperature. C. albicans grown at 30°C bound EAC3bi strongly, whereas those cells grown at 38.5°C were completely devoid of that feature. The C3d-binding proteins of C. albicans were purified by ion-exchange and affinity chromatography. Two proteins with molecular masses of 62 and 70 kDa were identified by silver stain, by Western blots (immunoblots) with CA-A, a monoclonal antibody against C. albicans that inhibited interaction of C. albicans with EAC3d, and by size exclusion high-pressure liquid chromatography (2, 3, 11). Ollert et al. (12) reported data on a human serum which inhibited the iC3b-mediated adherence almost completely; this serum reacted with 71-, 68-, 55-, and 50-kDa molecules in immunoblots of extracts C. albicans pseudohyphae. Here we report on the isolation of iC3b binding molecules from C. albicans. MATERIALS AND METHODS Reagents. OKM-1, a mouse monoclonal antibody of the immunoglobulin G2b (IgG2b) subtype directed against the alpha chain of human complement receptor type 3 (CR3, CD11b), was purchased from Ortho Diagnostic Systems, Raritan, N.J. C3, C3(H20), and the C3 fragments C3b, iC3b,

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SUMMARYIn this study we established a novel solid-phase immunoassay for CD21 using the time-resolved tluoresccnccof lanthanidechclates. The capture assay was able to detect concentrations ofas low as IOOpgofCD2l antigen per millil[trcor.saniple and was used for quantitative determination orCD21 in lysatcs of different cell lines as well as in patient serum specimens. CD21 was measured in lysates of tonsils and eell lines of B, T cell and myelomonoeyte lineage, and appeared to eonsist ofmonomeric aniigen under the detergent conditions used. Elevated levels of soluble CD2I were observed in serum of patients with Epstein Barr virus (EBV) infection, a disease known to be associated with polyclonal B cell activation, and in infection with ihe lymphotropie rubella virus. Significantly increased levels were also found in malignancies whieh are associated with EBV, In patients with nasopharyngeal carcinoma (NPC). a correlation with the litre of EBV-specific IgA was observed, thus supporting a possible role of soluble CD2I as a marker for disease activity in certain malignancies. Our data suggest that measurement of soluble CD21 could serve as a marker for activation of the immune system and diseases involving the B cell lymphoid system. Possible mechanisms and functions of soluble CD21 are discussed.

We studied the chemotactic effects of calcitonin gene-related peptide, vasoactive intestinal peptide, substance P (SP), and secretoneurin on PBMC and PBL using micropore filter assays. All four peptides induced migration of PBMC, whereas only calcitonin gene-related peptide, vasoactive intestinal peptide, and SP were chemotactic for PBL. Secretoneurin, known to induce monocyte chemotaxis, was unable to affect lymphocyte migration. Effects of SP on PBL were characterized by checkerboard analyses and represented true chemotaxis. Both T and B cells responded chemotactically to SP, the functional activity of SP residing in its C-terminal amino acid sequence. Involvement of neurokinin (NK) receptors was supported by inhibition of SP-induced migration of PBL with an NK1 receptor antagonist and induction of migration with [Sar9, Met(O2)11]SP and [PyrGlu6, Pro9]SP(6-11), two specific agonists for NK1 receptors, but not with [beta-Ala8]NK A(4-10), an agonist for NK2 receptors. PBL chemotaxis to SP was abolished by inhibition of tyrosin kinase but not by that of protein kinase C. Preincubation of PBL with pertussis or cholera toxin inhibited SP chemotaxis, indicating that in PBL, NK receptors for chemotaxis probably are coupled with G protein and involve a tyrosin kinase signaling pathway. We conclude that, together with calcitonin gene-related peptide and vasoactive intestinal peptide, SP is a lymphocyte chemoattractant, whereas secretoneurin, which is coreleased from sensory nerve endings, is not.