cDNA cloning and primary structure analysis of C1qR(P), the human C1q/MBL/SPA receptor that mediates enhanced phagocytosis in vitro.

The complement protein C1q, mannose-binding lectin (MBL), and pulmonary surfactant protein A (SPA) are structurally similar molecules that enhance phagocytic function in vitro. Monoclonal antibodies R3 and R139, which inhibit the enhancement triggered by these three ligands, were used to purify a 126,000 M(r) cell surface protein designated C1qR(P). Amino acid sequence was obtained and the corresponding cDNA was cloned. C1qR(P) is a novel type I membrane protein with the following putative structural elements: a C-type carbohydrate recognition domain, five EGF-like domains, a transmembrane domain, and a short cytoplasmic tail. All peptides identified by amino acid sequencing are encoded by the cDNA. Additionally, an anti-peptide antiserum was generated, which is reactive with C1qR(P). The data indicate that the cloned cDNA encodes the receptor that plays a role in C1q/MBL/SPA-mediated removal or destruction of pathogens and immune complexes by phagocytosis.

Biocompatibility of heparin-coated circuits used in cardiopulmonary bypass.

The combined effect of heparin coating of cardiopulmonary bypass (CPB) circuits and reduced dose of systemic heparin on activation of the complement system and blood leukocytes was investigated in 19 patients undergoing coronary bypass surgery and randomly allocated to two groups. A heparin-coated CPB circuit together with a 50% reduction of the standard heparin dose were used for ten patients (HC group), and a standard CPB circuit with a standard heparin dose (300 IU/kg) for nine (C group). Significant rise in the levels of neutrophil-derived myeloperoxidase, lactoferrin and calprotectin were observed during CPB in both groups, but the total accumulated levels were significantly lower in the HC than in the C group (p < 0.05). Complement activation, assessed from levels of C3a and terminal complement complexes was similar in both groups. The lower levels of myeloperoxidase, lactoferrin and calprotectin during CPB in the HC group indicate that surface modification with end-point attached heparin enhances the biocompatibility of CPB.

Reconstitution of murine resident peritoneal macrophages for antibody-dependent cellular cytotoxicity by homologous serum Clq.

Mouse resident peritoneal macrophages (PM) were reconstituted in their response to activation for antibody-dependent cellular cytotoxicity (ADCC) for sheep erythrocyte targets (SRBC) by subhemolytic dilutions of homologous or autologous sera. ADCC-responsive inflammatory PM were largely unaffected in their activation by exogenous serum. Augmentation of resident PM for ADCC by homologous serum was correlated with the complement-activating potential of the mouse monoclonal anti-SRBC IgG isotype in that serum augmented IgG gamma 2a greater than IgG gamma 2b much greater than IgG gamma 1. The active component of mouse serum was heat-labile at 56 degrees C for 30 min and was present in both C5-deficient AKR and C5-sufficient homologous C3H mouse sera. Western blot analysis of the cell lysates for Clq confirmed that oil-elicited and thioglycollate-elicited inflammatory PM had greater levels of endogenous Clq than did resident PM which correlated with their innate responsiveness for ADCC activation. Depletion of Clq from serum by immunoprecipitation with IgG antibody to Clq or by ion exchange chromatography removed the active reconstituting activity for ADCC. Purified mouse Clq (0.4 microgram) partially replenished the ADCC augmenting activity of Clq-depleted AKR mouse serum. SRBC targets preopsonized with IgG gamma 2a and purified mouse Clq (0.075-5.0 microgram/ml) fully reconstituted the ADCC response of resident PM similar to homologous serum indicating that the major active component of serum was Clq. Thus resident PM with low endogenous levels of Clq were reconstituted for ADCC by the addition of exogenous Clq, whereas inflammatory PM with sufficiently high endogenous levels of Clq were not further enhanced by exogenous Clq. Our findings indicate that Clq may provide an essential second signal in concert with Fc receptor binding of IgG to initiate ADCC activation of macrophages.

Association of levels of circulating C1q binding macromolecules with induction chemotherapy response in head and neck cancer patients.

Ninety-five untreated patients with squamous cell carcinoma of the upper aerodigestive tract expressed significantly higher levels of C1q-binding macromolecules as compared to 45 noncancer-bearing controls. No relationship between C1q-binding macromolecules and levels of circulating IgG-immune complexes as determined by the solid-phase C1q-binding assay or the C3d-solid-phase assay could be defined suggesting that C1q-binding macromolecules were distinct from IgG-circulating immune complexes. An elevated level of C1q-binding macromolecules within these patients was predictive of subsequent response to induction chemotherapy; those with elevated levels characteristically showed no response. Using multivariate logistic regression analysis including the covariates of American Joint Committee staging parameters as well as C1q assay results, levels of the isolated macromolecules added significant prognostic information as to the probability of chemotherapeutic response. The quantitation of C1q macromolecules has clinical implication as to choice of therapeutic regimens against head and neck cancer. The nature of these substances remains to be defined.

Use of glutaraldehyde-treated erythrocytes as indicator cells for the identification of the Clq component on the macrophage membrane by a rosette assay.

Glutaraldehyde-treated sheep red blood cells (EGA) form rosettes with macrophages of various origin. The binding of EGA to cell surface is mediated by the membrane associated Clq as demonstrated by the inhibition of EGA rosette formation either after treatment of macrophages with anti-Clq serum or after treatment of EGA with Clq. The results clearly demonstrate that the EGA-binding receptor of macrophage is the Clq component.

Glomerular annular-tubular immune deposits in adult hemolytic uremic syndrome.

An 82-year-old female developed hemolytic uremic syndrome (HUS) after a prodromal illness of bloody diarrhea. No specific enteric pathogen was isolated. A renal biopsy performed 5 days after the onset of azotemia revealed typical thrombotic microangiopathy. By electron microscopy, massive annular-tubular deposits admixed with fibrillar fibrin were demonstrated in glomerular capillaries. Immunofluorescent staining of the intracapillary material was positive for IgG, IgM, C3, C1q and fibrin-related antigens. No evidence of plasma cell dyscrasia, cryoglobulinemia or systemic lupus erythematosus was found, and the patient recovered renal function uneventfully in 2 months. Organized immune deposits appear to have played a role in the pathogenesis of HUS in this patient.

Characterization of immune complexes detected by the 125I-C1q binding assay in breast cancer.

The aim of the present study was to isolate and characterize immune complexes measured by the 125I-C1q binding assay in breast cancer sera. The C1q binding assay detects immune complexes by their binding to 125I-C1q and subsequent precipitation with PEG. We purified the C1q binding material from these precipitates by superose 6 gel filtration and cation exchange chromatography. The isolation steps were monitored by the C1q binding assay and the purified material was analyzed by SDS--PAGE and immunodiffusion. The C1q binding material isolated from two breast cancer sera contained complexes of IgM, C4b binding protein (C4-bp), and C1q. The C4-bp fraction showed significant C1q binding activity which increased after the addition of IgM. This C4-bp was not different from C4-bp isolated from normal serum. The IgM fraction, however, differed from normal IgM by its binding to C4-bp and C1q and its stronger affinity to the cation exchange column. These unique properties were not due to rheumatoid factor activity. They might be caused by a different glycosylation pattern or by complex formation with an as yet unknown polysaccharide antigen.

Deposits of immunoglobulins, complement, and immune complexes in inflamed human gingiva.

Gingival biopsy specimens from 20 patients with moderate to advanced periodontitis were obtained from inflamed sites with pockets of 5 mm or more. Sections were studied by an immunofluorescence technique, using polyclonal rabbit or goat anti-IgG, anti-IgM, anti-C1q, anti-C3a, and anti-C3c and mouse monoclonal anti-C9. Prewashed ethanol-fixed and nonwashed ethanol-fixed or frozen specimens showed many plasma cells staining for IgG or C3a, suggesting the possible occurrence of a receptor for C3a in plasma cells. Plasma cells containing IgM were also seen. Deposits of IgG and IgM with C1q, C3a, and C3c, suggesting immune complexes, were demonstrated by a double staining technique, combining fluorescein (FITC) or rhodamine (TRITC)-labeled anti-immunoglobulins with TRITC- or FITC-conjugated antibody to C3a, C3c, and C1q. The complexes were located mainly within or around vessel walls. Deposits of C3a and C1q were found in vessel walls, in the basement membrane zone of oral gingival epithelium, or diffusely distributed in the tissues. Deposits of C3c were found to a lesser extent and only in vessel walls. Mouse monoclonal anti-C9, visualized with FITC-labeled rabbit anti-mouse and swine anti-rabbit antiserum, showed granular deposits of C9, mainly in the basement membrane zone of oral gingival epithelium. The study indicates the involvement of immune complex vasculitis in inflammatory periodontal lesions. Also, our observations of the occurrence of deposits of complement factors support the hypothesis that complement factors play an important role in the immunopathology of the periodontal lesion.

Relationship between renal pathology and the size of circulating immune complexes in patients with systemic lupus erythematosus.

Sera from 35 patients with biopsy-proven diffuse proliferative (WHO class IV) or membranous (WHO class V) lupus nephritis were analyzed for the presence and size of circulating immune complexes. Elevations of the C1q solid-phase assay (C1qSP) for immune complexes were found in sera from all patients with diffuse proliferative nephritis, with a mean +/- 1 SEM of 166.8 +/- 42.0 micrograms/AHG-equivalents/ml serum, and in 71.4% of the patients with membranous nephritis (83.1 +/- 26.7, p = 0.06). Using the WHO criteria for subclasses of membranous lupus nephritis, we also designated renal biopsies as nonproliferative (WHO classes Va and Vb) or proliferative (WHO classes IV and Vc). Employing the latter groupings, we observed significant differences between C1qSP results of patients with nonproliferative (30.3 +/- 8.8) and proliferative (172.8 +/- 36.8, p less than 0.001) lupus nephritis. These data suggest that the presence of C1q-binding material in serum is pathophysiologically related to proliferative glomerular lesions, and that levels of C1qSP binding reflect renal lesions in SLE patients. Sucrose density gradient ultracentrifugation was performed on each serum, and gradient fractions analyzed for C1qSP-binding and total IgG, using techniques to minimize losses of immune complexes. The predominant peak of C1qSP activity sedimented with the 6.6S monomeric IgG. The 6.6S C1q-binding IgG was increased only in 1 of 10 patients with membranous lupus nephritis without proliferative changes, and was elevated in 16 of 25 patients with proliferative lesions (WHO classes IV and Vc). A significant negative correlation was found between the presence of this C1q-binding material and subepithelial electron-dense deposits, suggesting that the presence of this material contributed to the absence of subepithelial immune deposits. Large-molecular-weight C1qSP-binding material was also present, mainly in sera from patients with proliferative lesions. Furthermore, highly positive correlations were found between immune deposits in interstitial blood vessels and peritubular areas, and the concentrations of C1qSP-binding IgG and rapidly sedimenting IgG in density gradient analysis. Overall, these findings are consistent with the hypotheses that circulating immune complexes contribute to the pathogenesis of glomerulonephritis and interstitial nephritis in patients with SLE, and that 6.6S C1q-binding IgG plays a role in the proliferative lesions of lupus glomerulonephritis.

Dense deposit disease: its possible pathogenesis suggested by an observation of a patient.

A girl, aged 8, was admitted to a hospital in a state of nephrotic syndrome of one year's duration. The renal biopsy showed mesangial and endocapillary proliferations with lobulation. Dense deposit was not demonstrated by electron microscopy, but lamellation of the lamina densa was found in most of the capillary loops. Her condition was improved by steroid treatment in a few months, but moderate proteinuria persisted. Five and half years later, follow-up biopsy showed typical pathological features of dense deposit disease. It is suggested that the lamellation of the lamina densa in the first biopsy could be related to the dense alteration of glomerular basement membrane in the second biopsy.

Detection of C-reactive protein, streptolysin O, and anti-streptolysin O antibodies in immune complexes isolated from the sera of patients with acute rheumatic fever.

Circulating immune complexes (IC) of 42 patients with acute rheumatic fever from Santiago, Chile, were studied. The complexes were isolated by polyethylene glycol precipitation and were analyzed for antibodies, antigens, and C-reactive protein. We found the complexes to be enriched in antibody to streptolysin O, particularly in the group of patients with elevated levels of IC. IgM was the predominant class of Ig present in the complexes. Western blots from 12 patients to detect antigens in the complexes showed proteins of m.w. 50,000, 60,000, and 69,000, consistent with the polypeptides of streptolysin O. Such antigens were absent in the complexes from patients with post-streptococcal glomerulonephritis and pharyngitis. Eluted antibodies from these protein bands on the nitrocellulose sheets reacted with the streptolysin O in Western blots and neutralized the hemolytic activity of streptolysin O in a microhemolysin assay. In addition, isolated complexes from several sera showed the presence of C-reactive protein bound to complexes. In vitro experiments demonstrated that [125I]C-reactive protein was not precipitated by polyethylene glycol either alone or when added to monomeric IgG, whereas it precipitated significantly when added to aggregated IgG. The detectable C-reactive protein in isolated complexes and sera samples increased after treatment with sodium dodecyl sulfate. These data suggest that circulating immune complexes in acute rheumatic fever contain streptolysin O and its antibody and raise interesting questions regarding the pathogenetic significance of C-reactive protein in the complexes.

Changes of serum complement concentrations in tumour patients prior and after radiotherapy.

Peripheral blood concentrations of leucocytes, platelets and complement factors Clq, C3c, C3d, C4 and C5 were examined in 30 patients suffering from malignant tumours prior and after radiotherapy. In general, a decrease of blood cell concentrations as well as serum complement levels was noted using regression analysis. Two groups were formed: patients with (group I) and without (group II) foregoing tumour surgery. A positive correlation was found for all complement factors prior therapy in group I, whereas only some complement components did correlate in group II. The results in group I are in agreement with complements classical pathway activation. We conclude that the alternative pathways influence is responsible for the different results in group II.

Unusual dermatologic toxicity of long-term therapy with hydroxyurea in chronic myelogenous leukemia.

The unusual appearance of extensive skin ulcerations was observed in 17 patients with chronic myelogenous leukemia on continuous chemotherapy with hydroxyurea. The strict relationship between ulcers and therapy was proved by the complete (14 cases) or almost complete (3 cases) healing of lesions after therapy was discontinued. The possible pathogenetic mechanisms responsible for skin alterations are considered. The particular importance of the continuous hydroxyurea administration modality clearly emerges, suggesting the use of different administration modalities to reduce such serious side effects.

Primary idiopathic cutaneous pustular vasculitis.

Pustular cutaneous vasculitis results from a heterogeneous group of disorders characterized by pustules on purpuric bases. Although the cause of this group of conditions is diverse, the histopathologic picture of the lesions is the same, showing a Sweet's-like or leukocytoclastic vasculitis. These distinctive lesions may occur in patients with Behçet's syndrome, bowel-associated dermatosis-arthritis syndrome, or chronic gonococcemia. We describe, for the first time, a patient with primary idiopathic cutaneous pustular vasculitis. This patient had evidence of both circulating immune complexes and serum enhancement of neutrophil migration. Extensive evaluation failed to reveal any underlying systemic disease. A classification of the pustular vasculitides is proposed.

Circulating immune complexes in Meniere's disease.

Sixty-six patients with Meniere's disease were studied for possible general immunologic abnormalities. The disease was bilateral in 20 cases, causing active symptoms in 49. Thirty-six healthy control subjects were also studied. Immune activity was assessed by quantitative measurement of the circulating immune complexes, serum immunoglobulin assay, and acetate electrophoresis, and by an autoantibody screen to a battery of ten general tissue antigens. Thirty-six (54.5%) of the 66 patients with Meniere's disease were found to have significantly raised circulating immune complex levels compared with one (2.9%) of the 36 controls. A statistically significant increased incidence of autoantibodies was also found in the Meniere's group.

Characterization of the antigenic determinants and host components in immune complexes from patients with secondary syphilis.

Sera from patients with secondary syphilis were evaluated for abnormal levels of circulating immune complexes (IC), immunoglobulins (Ig), and complement components. Clq-solid-phase assays (Clq-SPA) that made use of monoclonal and polyclonal antibodies directed against IgG subclasses indicated that human IC were composed primarily of IgG3 and IgG1; these findings appeared consistent with subclass profile responses of electrophoretically transferred blots (Western blots) of Treponema pallidum reacted with syphilitic sera. Complexes were isolated from reactive sera by polyethylene glycol precipitation followed by either anti-Clq column chromatography or protein A-Sepharose chromatography. Although qualitative and quantitative differences were noted, all purified materials contained a treponemal polypeptide antigen with a m.w. of approximately 87,000. Subsequent analysis of this polypeptide, which was also present in purified IC from rabbits with experimental syphilis, suggests that it may represent the fibronectin receptor of the organism. The 76,000 and 66,000 materials, earlier identified in purified rabbit IC, appeared to represent C-terminal degradation products of fibronectin presumably of host origin, rather than treponemal antigens. Although fibronectin binds avidly to Clq and could represent a co-precipitable contaminant throughout the isolation procedure, anti-fibronectin antibodies in the sera of patients detectable by radioimmunoassay and the present of antibodies to 76,000 and 66,000 dalton fibronectin fragments in the globulin fractions of disassociated complexes argues against such a conclusion.