Complement-immunoglobulin relation: deficiency of C'1q associated with impaired immunoglobulin G synthesis.

Concentration of the complement protein C'lq was determined immunochemically in serums of individuals with a wide variety of immunoglobulin abnormalities. A significant correlation was observed between decreased concentration of C'lq and deficient synthesis of immunoglobulin G; C'lq was particularly diminished in subjects with congenital, sex-linked (Bruton) agammaglobulinemia. In contrast, two to five times the normal concentration of C'lq was found in the serum of three patients with heavy chain disease (subtype immunoglobulin G3). No significant relation was found between C'lq and concentrations of immunoglobulins A and M.

Maturation of the human complement system. I. Onset time and sites of fetal C1q, C4, C3, and C5 synthesis.

The onset times and sites of human C1q, C4, C3, and C5 synthesis were determined by culturing tissues from 23 fetuses, 8-25 wk old, in the presence of [(14)C]lysine and isoleucine. In parallel, IgG and IgM production was followed. Liver, spleen, placenta, peritoneal and bone marrow cells, thymus, and colon were cultured for 48 h and the concentrated media studied by immunoelectrophoresis and subsequent autoradiography using adult human serum as carrier and specific antisera. The quantitative synthesis was approximated by scoring the intensity of the labeled precipitin lines using uniform conditions. C5 production was detected earliest at 8 wk gestation and by 11 wk and thereafter, C3, C4, and C5 synthesis was uniformly present in multiple tissues. C1q synthesis, however, was limited almost exclusively to the spleen, began at 14 wk, and was not uniformly present. In contrast IgG and IgM production did not occur in three fetuses synthesizing complement and while detected as early as 11 wk. was inconstant, occurred predominantly in the spleen, and was quantitatively much less compared to C3, C4, and C5. These findings suggest that developmentally the complement system is a more primative biological defense mechanism than antibody.

Metabolism of human C1q. Studies in hypogammaglobulinemia, myeloma, and systemic lupus erythematosus.

The in vivo metabolism of radioiodinelabeled C1q was determined in patients with hypogammaglobulinemia, multiple myeloma, systemic lupus erythematosus (SLE), and in healthy controls. Marked differences in metabolic behavior were observed with a much more rapid disappearance of plasma radioactivity in patients as compared with controls. Estimated plasma volumes at 10 min after injection (time 0) were normal in controls and the SLE patient, mean 40 ml/kg, whereas they were grossly elevated, 57-82 ml/kg, in the hypogammaglobulinemic and myeloma patients, indicating significant loss of C1q-(125)I during the initial mixing period. Absence of a distinct initial equilibration phase of radioactivity loss from the plasma suggested significant reversible interaction of the labeled C1q with plasma proteins and density gradient studies provided evidence for in vivo uptake into the circulating trimolecular first component complex (C1q, r, s). In controls and the SLE patient 0.51-0.75 of the C1q was retained in the plasma space while only 0.28 or less was in the others. The daily plasma pool fractional C1q catabolism was 0.65-0.67 in controls compared with 0.95-4.80 in the patients. C1q synthetic rates in controls were 4.64 and 4.34 mg/kg per day while higher rates, 4.94-37.40 occurred in the patients. These experiments clearly indicate that the metabolism of C1q is markedly influenced by serum IgG concentrations, probably related to the reversible interactions of C1q with IgG, and also affected by interactions with C1r and C1s. The decreased serum C1q often present in hypogammaglobulinemia and myeloma relates to an increased catabolism and higher extravascular distribution rather than impaired C1q synthesis. In contrast, a second distinctly different basis for decreased C1q occurs in SLE; increased utilization by an ongoing immunopathogenic process.

Human monoclonal gamma G-cryoglobulins with anti-gamma-globulin activity.

Seven human gammaG-myeloma proteins which were also cryoglobulins were studied with respect to their reactivity with gammaG-globulins as well as with regard to their antigenic classification within the gammaG-heavy chain subclasses. Five of the seven cryoglobulins studied were positive in at least two of the three tests used to assay for anti-gamma-globulin activity. One protein was only weakly positive in one test system and another was negative in all test systems. The structures which were recognized by the cryoglobulins were localized to the Fc-fragment. Only primate gammaG-globulins contained these antigenic determinants and in some cases the cryoglobulin appeared to show specificity for one human heavy chain subclass over the others. Antigenic analysis revealed that four of the five cryoglobulins with definite antibody activity belonged to the gammaG3-subclass, the fifth belonged to the gammaG1-subclass. The two cryoglobulins which reacted only weakly or failed to combine with gammaG-globulins were both of the gammaG1-subclass. These findings taken together with the localization of the combining site to the Fab-fragment suggests that many of these cryoglobulins may represent antibodies to gammaG-globulin, and that the cryoprecipitate in these cases represents antigen-antibody complexes of such a nature that they precipitate only in the cold.

DNA:anti-DNA complexes: their detection in systemic lupus erythematosus sera.

Antibody to DNA was measured before and after treatment of systemic lupus erythematosus (SLE) sera with bovine pancreatic deoxyribonuclease (DNase I). In 11 of 15 cases of SLE with active renal disease there was a significant increase in DNA-binding after DNase digestion, while no such increase was noted in inactive SLE, normal controls or in patients with nonlupus renal disease. The significant rise in DNA-binding after digestion indicated that DNA had bound in vivo to the anti-DNA in these sera. A striking correlation between the occurrence of these complexes and disease activity was shown. In eight cases of SLE nephritis where serial blood samples were obtained, the greatest increase in DNA-binding after DNase digestion occurred at the time of the severest renal disease. In addition, serum from a case of SLE with acute cerebritis but without evidence of renal disease also had a significant rise in binding during the acute phase. This assay provides proof of the existence of circulating DNA:anti-DNA complexes in some cases of SLE and can also be used to measure an apparently critical parameter of disease activity.

Genetic regulation of immunoglobulin and specific antibody levels in twins reared apart.

We studied the effect of the same genetic but different environmental factors on total immunoglobulin and specific antibody levels in twins reared apart. Sera were analyzed from 26 monozygotic (MZ) and 10 dizygotic (DZ) twin pairs, who were separated on average 2 mo after birth and reared apart. Total IgM, IgG, and IgA were measured by single radial diffusion. Specific antibodies of each isotype to tetanus toxoid, and to polyvalent and type 14 pneumococcal capsular polysaccharides were measured by a solid-phase antigen-enzyme-labeled anti-Ig immunoassay. One-way analysis of variance showed intrapair total Ig and antibody levels to be more highly correlated in MZ compared with DZ twins. Our results indicate that genetic factors are more important than environment in regulating these humoral immune responses.

Meningococcemia and acquired complement deficiency. Association in patients with hepatic failure.

We treated two patients with severe hepatic failure complicated by meningococcemia. Serum complement profiles performed on these patients found low total hemolytic complement assays, normal concentrations of C1q, and low or undetectable concentrations of C3 through C6, C8, C9, and factors B and I. These studies suggest that these patients developed meningococcemia in the setting of acquired complement deficiency from impaired synthesis of multiple complement system proteins.

Acute nephritis and pulmonary alveolitis following pneumococcal pneumonia.

Acute glomerulonephritis developed in a man with pneumococcal pneumonia. Serum complement studies revealed decreased levels of C4, properdin, and C3. Renal immunofluorescence studies demonstrated pneumococcal antigen, C1q, C4, C3 proactivator, properdin, C3, IgG, and IgM. Circulating cryoglobulin contained pneumococcal antigen and antibody, C3, and immunoglobulins. Serial pneumococcal antigen and antibody levels did not display patterns that were characteristic of classical immune elimination, but the patterns may have been influenced by the reentry of antigen. A diffuse, pulmonary alveolitis also developed in the patient. Lung immunofluorescence studies revealed pneumococcal antigen, IgG, and C3 in alveolar walls and capillary basement membranes. The glomerulonephritis and alveolitis resolved after a prolonged course. These findings provide presumptive evidence for pneumococcal, immune complex glomerulonephritis with complement activation via both classical and alternative pathways and suggest an immunologic pathogenesis for the pulmonary alveolitis.

Results of the National Institute of Allergy and Infectious Diseases Collaborative Clinical Trial to test the predictive value of skin testing with major and minor penicillin derivatives in hospitalized adults.

BACKGROUND: A history (or lack thereof) of penicillin allergy is known to be unreliable in predicting reactions on subsequent administration of the drug. This study tests the usefulness of four penicillin allergen skin tests in the prediction of IgE-mediated reactions subsequent to administration of penicillin. METHODS: Eight centers cooperated in the National Institute of Allergy and Infectious Diseases trial of the predictive value of skin testing with major and minor penicillin derivatives. Hospitalized adults were tested with a major determinant (octa-benzylpenicilloyl-ocytalysine) and a minor determinant mixture and its components (potassium benzylpenicillin, benzylpenicilloate, and benzylpenicilloyl-N-propylamine). Patients then received a therapeutic course of penicillin and were observed, for 48 hours, for adverse reactions compatible with an IgE-mediated immediate or accelerated allergy. RESULTS: Among 726 history-positive patients, 566 with negative skin tests received penicillin and only seven (1.2%) had possibly IgE-mediated reactions. Among 600 history-negative patients, 568 with negative skin tests received penicillin and none had a reaction. Only nine of the 167 positive skin test reactors received a penicillin agent and then usually by cautious incremental dosing. Two (22%) of these nine patients had reactions compatible with IgE-mediated immediate or accelerated penicillin allergy; both were positive to the two determinants. CONCLUSIONS: These data corroborate previous data about the negative predictive value of negative skin tests to these materials. The reaction rate in skin test-positive patients was significantly higher than in those with negative skin tests, demonstrating the positive predictive value of positive tests to both major and minor determinants. The number of patients positive only to the major determinant or only to the minor determinant mix was too small to draw conclusions about the positive predictive value of either reagent alone.

Transcutaneous leukocyte migration in vivo: cellular kinetics, platelet and C5a dependent activity.

A simple quantitative method for the measurement of leukotaxis in vivo is described. Duplicate skin chambers are placed over tape-stripped skin with 50% autologous serum--50% Hank's balanced salt solution as the attractant. Neutrophils predominate throughout 24 hr in this method with no change to mononuclear cells. A recommended modification of our original method is that chambers are sampled and removed after 8 hr rather than 24 hr since the majority of leukocyte migration occurs within the first 8 hr. Analysis of serum factors showed that heat-inactivation of the serum (56degreesC for 30 min) had no effect. However, depleting platelets or C5 from the serum removed approximately 90% of chemotactic activity for human neutrophils in vivo. Platelets, presumably through activity of their granules, enzymatically cleaves C5a from C5 in plasma. We conclude that C5a, after cleavage from C5, accounts for the majority of chemattractant activity in vivo.

Leukocytoclastic vasculitis: sequential appearance of immunoreactants and cellular changes in serial biopsies.

To study the mechanisms responsible for leukocytoclastic vasculitis, we evaluated the kinetics of immunologic and cellular changes in induced vasculitis lesions. In four of five consecutive patients with active vasculitis, lesions were induced by increasing vascular permeability via injecting histamine into the skin. Biopsies were obtained for light and electron microscopy and immunofluorescence at 1, 4, 8, and 24 hr after injection. The results show that immunoglobulin, C3, and electron-dense material are deposited in vessel walls early and are followed by cellular infiltration. The characteristics of the cellular infiltrates were quite diverse at different times after histamine provocation and no distinctive patterns were seen. Nevertheless, the kinetics of the appearance of immunoreactants and cells implies that immunoglobulin and probably circulating immune complexes are present prior to the development of inflammation and supports the contention that deposition of immune complexes within vessel walls is responsible for leukocytoclastic vasculitis.

Human necrotizing vasculitis: immunoglobulins and complement in vessel walls of cutaneous lesions and normal skin.

Immunoglobulins and C3 were detected by immunofluorescence in the blood vessel walls of biopsies of clinically normal skin in patients with active necrotizing vasculitis. Of the 13 patients studied, 9 had C3 and 6 of these had IgM or IgA in biopsies of lesions of vasculitis. In adjacent clinically normal skin, 7 patients had C3 and 3 of these also had IgM or IgA. These findings support the hypothesis that immunoglobulins and complement are present in vessels of some patients prior to chemotaxis of polymorphonuclear leukocytes and the resulting inflammatory purpuric lesions so characteristic of necrotizing vasculitis.

Leptin in anorexia nervosa.

Serum leptin levels are low in untreated anorexia nervosa, but studies of the exact relationship between leptin and body weight and the impact of refeeding in anorectics are limited. Therefore, we studied serum leptin, insulin-like growth factor I, and other endocrine parameters in female anorectics before and after gaining weight and in female normal body weight controls. Leptin levels in untreated anorectics were significantly lower than those in normal body weight controls (3.6 +/- 1.6 vs. 12.0 +/- 6.9 ng/mL; P < 0.001), and they uncoupled from body weight in a nonlinear relationship, suggesting a threshold effect at lowest body weights. Leptin increased significantly with refeeding (5.6 +/- 3.8 ng/mL; P < 0.01). The significant linear correlations of leptin with body mass index in the anorectics after weight gain and in normal body weight controls (r = 0.69; P < 0.001 and r = 0.76; P < 0.001, respectively) are consistent with a normal physiological increase in leptin with weight gain. Leptin and insulin-like growth factor I were highly correlated, even after controlling for body weight (r = 0.63; P = 0.001) during starvation, but were no longer significantly correlated after body weight gain in the anorectics or the normal body weight controls. Further studies are necessary to elucidate the relationship of leptin to neuroendrocrine abnormalities seen in starvation and to determine a possible contribution of leptin to difficulties with weight restoration in anorexia nervosa.

Double fluorescence technique for measurement of complement-fixing antibody to lymphocyte subsets.

A double fluorescent antibody method for quantitating human complement-fixing antibody to lymphocyte subclasses has been developed. The indicators in this system are a C6-deficient serum as a non-lytic source of complement, rhodamine-labeled anti-C3 and fluorescein-labeled murine monoclonal antibodies to human lymphocyte subsets. The basic procedure is to incubate lymphocytes with the unknown serum and then to add C6-deficient serum. The binding of C3 is indicated by staining with rhodamine-labeled anti-C3 and the subset class of the lymphocyte so stained is determined by binding of fluorescein-tagged anti-OKT4 or -OKT8 antibodies. The occurrence of both red and green cell surface fluorescence denotes the presence of a complement-binding antibody to the lymphocyte subset defined by the monoclonal antibody. In addition to defining the specificity of complement-fixing anti-lymphocyte antibodies, this technique is more sensitive than the microcytotoxicity assay.

Onset of polyarteritis nodosa during allergic hyposensitization treatment.

In six of 20 consecutive patients with polyarteritis nodosa, the onset of vasculitic symptoms coincided with hyposensitization therapy for presumptive atopic (immunoglobulin E-mediated) respiratory disease. Atopic symptoms had been present for less than three months in half of the patients and over 10 years in the remainder. Active vasculitis persisted in all patients despite immediate cessation of the hyposensitization treatment. Three patients died within eight months. When compared with the 14 other patients with polyarteritis nodosa, those undergoing hyposensitization had significantly greater skin involvement and peripheral blood eosinophilia (p = less than 0.05). Evidence for circulating immune complexes with decreased hemolytic complement, increased cryoglobulins or increased Clq binding was present in both groups. No single allergen was used in all patients, no antiallergen precipitating antibodies were detected and less than 16 mg of allerginic protein had been injected in five of the patients.

Antibodies against retroviral proteins and nuclear antigens in a subset of idiopathic CD4+ T lymphocytopenia patients.

Idiopathic CD4+ T lymphocytopenia (ICL) is an immunodeficiency syndrome characterized by severe depletion of CD4+ T lymphocytes, but in which human immunodeficiency virus cannot be detected. Peripheral blood mononuclear cells (BPMCs) from an ICL patient were cocultured with HUT78 T-lymphoblastoid cells, and an acute cytopathic effect and formation of multinucleated cells were observed. A human intracisternal A-type retroviral particle designated HIAP-II was detected in cells surviving the acute cytopathic effect. Eight of 13 ICL patients in a blinded screen of a serological panel provided by the National Centers for Disease Control and Prevention (CDC) had serum antibodies that specifically reacted with HIAP-II associated proteins by Western immunoblotting. None of 19 control sera in the panel that were unreactive with HIV Gag proteins produced a positive result on HIAP-II immunoblots. Comparable results were obtained in a blinded screen of a second CDC serological panel. Sera from 8 of 14 ICL patients in the second serological panel were positive for antinuclear autoantibodies (ANAs) commonly observed in patients with systemic autoimmune diseases. These results suggest the possible involvement of an A-type retrovirus or autoimmunity in development of ICL in a subset of patients.

Hypersensitivity pneumonitis and extrinsic asthma. An unusual association.

A farmer who had no prior history of pulmonary disease developed tightness in the chest of rapid onset, shortness of breath, fever, and pulmonary infiltration while farming. The symptoms of his disease worsened with repeated exposure to the dusty farm field but remitted after each of five hospitalizations. Provocative challenge with inhalation of a water-soluble extract of dust from the field reproduced both asthmatic and pneumonitic features of the disease, while administration of corticosteroids clinically controlled the entire process. The data suggest a common cause for asthma and pneumonitis in this patient.

Delayed pressure urticaria histologically resembles cutaneous late-phase reactions.

In a recent study, all patients with delayed pressure urticaria (DPU) developed late cutaneous reaction (LCR) after intradermal injection of compound 48/80 and after skin testing with certain food antigens. In the present study, we analyzed the histologic changes in the pressure lesions and compared them with those found in normal skin injected with diluent and in LCR to 48/80. The study included five patients with DPU associated with chronic urticaria (CU) and four patients with CU but without DPU. Six to eight hours after pressure challenge and intradermal skin testing with 48/80 and diluent, skin biopsy specimens were obtained from the pressure lesions, the LCRs, and normal skin (diluent injection). Specimens were assessed by Giemsa staining of 1-micron sections and immunofluorescence of frozen sections. Total cells were counted in each specimen. Interstitial deposits of fibrin were observed by immunofluorescence in LCR and pressure lesions. The total numbers of infiltrating cells in the dermis among LCR sites and pressure lesions were not significantly different, while both LCR sites and pressure lesions contained significantly more infiltrating cells than did normal skin injected with saline diluent. The differential counts in LCR and DPU were mostly mononuclear cells. Infiltrates in the DPU and LCR were mostly perivascular. No histopathologic changes were seen at skin sites challenged with pressure in the control patients with CU without clinical manifestations of DPU. We conclude that lesions seen in DPU are morphologically similar to classic LCR.