Absence of diurnal variation of C-reactive protein concentrations in healthy human subjects

BACKGROUND: The concentration of C-reactive protein (CRP) in otherwise healthy subjects has been shown to predict future risk of myocardial infarction and stroke. CRP is synthesized by the liver in response to interleukin-6, the serum concentration of which is subject to diurnal variation. METHODS: To examine the existence of a time-of-day effect for baseline CRP values, we determined CRP concentrations in hourly blood samples drawn from healthy subjects (10 males, 3 females; age range, 21-35 years) during a baseline day in a controlled environment (8 h of nighttime sleep). RESULTS: Overall CRP concentrations were low, with only three subjects having CRP concentrations >2 mg/L. Comparison of raw data showed stability of CRP concentrations throughout the 24 h studied. When compared with cutoff values of CRP quintile derived from population-based studies, misclassification of greater than one quintile did not occur as a result of diurnal variation in any of the subjects studied. Nonparametric ANOVA comparing different time points showed no significant differences for both raw and z-transformed data. Analysis for rhythmic diurnal variation using a method fitting a cosine curve to the group data was negative. CONCLUSIONS: Our data show that baseline CRP concentrations are not subject to time-of-day variation and thus help to explain why CRP concentrations are a better predictor of vascular risk than interleukin-6. Determination of CRP for cardiovascular risk prediction may be performed without concern for diurnal variation.     

Binding of immunoglobulin G aggregates and immune complexes in human sera to Staphylococci containing protein A.

Using the Cowan I strain of Staphylococcus aureus, we compared the binding properties of human monomeric immunoglobulin (Ig)G and oligomeric or complexed IgG. Heat-aggregated IgG served as a model for complexed IgG and heat-killed, formalin-fixed S. aureus (StaphA) as a cellular receptor for IgG, in determining the parameters for oligomeric and monomeric binding. Because of its capacity for multipoint attachment, complexed IgG binding was favored over monomeric IgG binding, and this preferential binding was demonstrated kinetically in equivalent forward rates of binding but in a much slower rate of release from StaphA receptors. From binding studies, we determined which conditions maximize complexes IgG binding and minimized monomeric IgG binding and applied them to the development of an assay for aggregated IgG and immune complexes in human sera. The StaphA binding assay that was devised is quantitative, sensitive, and not complement dependent. It is relatively unaffected by factors such as heparin, complement fixation, native antibodies, and immunoglobulin concentrations, but is affected by the presence of rheumatoid factors. It compares favorably with two other complement-dependent assays of immune complexes, the 125I-Clq binding assay and the Raji cell assay, in terms of sensitivity and the size of immune complexes detected. Studies on the potential of the assay for detecting, isolating, and characterizing immune complexes in biological fluids are presented.     

Presentation of antigen in immune complexes is boosted by soluble bacterial immunoglobulin binding proteins

Using a snake toxin as a proteic antigen (Ag), two murine toxin-specific monoclonal antibodies (mAbs), splenocytes, and two murine Ag-specific T cell hybridomas, we showed that soluble protein A (SpA) from Staphylococcus aureus and protein G from Streptococcus subspecies, two Ig binding proteins (IBPs), not only abolish the capacity of the mAbs to decrease Ag presentation but also increase Ag presentation 20-100-fold. Five lines of evidence suggest that this phenomenon results from binding of an IBP-Ab-Ag complex to B cells possessing IBP receptors. First, we showed that SpA is likely to boost presentation of a free mAb, suggesting that the IBP-boosted presentation of an Ag in an immune complex results from the binding of IBP to the mAb. Second, FACS analyses showed that an Ag-Ab complex is preferentially targeted by SpA to a subpopulation of splenocytes mainly composed of B cells. Third, SpA-dependent boosted presentation of an Ag-Ab complex is further enhanced when splenocytes are enriched in cells containing SpA receptors. Fourth, the boosting effect largely diminishes when splenocytes are depleted of cells containing SpA receptors. Fifth, the boosting effect occurs only when IBP simultaneously contains a Fab and an Fc binding site. Altogether, our data suggest that soluble IBPs can bridge immune complexes to APCs containing IBP receptors, raising the possibility that during an infection process by bacteria secreting these IBPs, Ag-specific T cells may activate IBP receptor-containing B cells by a mechanism of intermolecular help, thus leading to a nonspecific immune response.     

C-reactive protein binding to FcgammaRIIa on human monocytes and neutrophils is allele-specific

C-reactive protein (CRP) is involved in host defense, regulation of inflammation, and modulation of autoimmune disease. Although the presence of receptors for CRP on phagocytes has been inferred for years, their identity was determined only recently. FcgammaRIa, the high-affinity IgG receptor, binds CRP with low affinity, whereas FcgammaRIIa, the low-affinity IgG receptor, binds CRP with high affinity. Because the single nucleotide polymorphism in FcgammaRIIA - which encodes histidine or arginine at position 131 - strongly influences IgG2 binding, we determined this polymorphism's effect on CRP binding. CRP bound with high avidity to monocytes and neutrophils from FcgammaRIIA R-131 homozygotes, and binding was inhibited by the R-specific mAb 41H16. CRP showed decreased binding to cells from FcgammaRIIA H-131 homozygotes (which bind IgG2 with high affinity). However, IFN-gamma enhanced FcgammaRI expression by H-131 monocytes and increased CRP binding. FcgammaRIIa heterozygotes showed intermediate binding. CRP initiated increases in [Ca(2+)](i) in PMN from R-131, but not from H-131 homozygotes. These data provide direct genetic evidence for FcgammaRIIa as the functional, high-affinity CRP receptor on leukocytes while emphasizing the reciprocal relationship between IgG and CRP binding avidities. This counterbalance may affect the contribution of FcgammaRIIA alleles to host defense and autoimmunity.     

The detection of circulating immune complexes containing immunoglobulin G

An immune complex assay using radiolabelled immunospecific antibodies against human IgG and polyethylene glycol precipitation (IgG-PEG assay) is described. The material reactive in this assay was evaluated using aggregated immunoglobulins, immune complexes prepared in vitro, sera of patients with a variety of disorders and normal human serum. Sucrose density gradient ultracentrifugation showed that only large-sized immune complexes (greater than 25 S) were detected. Comparison of the results of the IgG-PEG assay with those of the C1q binding assay showed a highly significant positive correlation (p less than 0.001). It was found that rheumatoid factors do not influence the results of the IgG-PEG assay. The method described in this study detects specifically immune complexes containing IgG and might be extended to the detection of other constituents of circulating immune complexes.     

Differential binding of immunoglobulin A and immunoglobulin G1 immune complexes to primate erythrocytes in vivo. Immunoglobulin A immune complexes bind less well to erythrocytes and are preferentially deposited in glomeruli.

Primate erythrocytes appear to play a role in the clearance of potentially pathogenic immune complexes (IC) from the circulation. This study was undertaken to compare the clearance from the circulation and tissue uptake of two monoclonal IC probes: one of which, IgG1-IC, was bound well by erythrocytes, the other of which, IgA-IC, was bound relatively poorly by erythrocytes. The IC probes were labeled with different iodine isotopes and infused either concomitantly or sequentially into the arterial circulation. The results indicate that, compared with IgG1-IC, IgA-IC bind less well to primate erythrocytes, are cleared from the circulation more quickly despite their smaller size, and show increased uptake in kidney and lung but decreased uptake in liver and spleen. Evidence is presented which suggests that this pattern of clearance from the circulation and systemic uptake of IgA-IC is the result of decreased binding of IgA-IC to circulating erythrocytes. These findings support the hypothesis that the primate erythrocyte-IC clearing mechanism may be critically important for the safe removal of IC from the circulation.     

Significance of C-reactive protein binding by lecithin: a simplified procedure for CRP isolation

Addition of lecithin to whole serum or liver extract in the presence of calcium ions resulted in the binding of C-reactive protein (CRP) to lecithin with formation of floccules. Subsequent treatment of the CaCl₂—distilled water washed lecithin—CRP complexes following suspension in citrate—saline with chloroform resulted in the isolation of essentially purified CRP preparations. The major serum component precipitating with CRP was immunoglobulin M, which was found in 3 of 9 CRP preparations. Yields ranged from 43.7 to 89.1% recovery of CRP from original pooled acute phase serum samples. Both γ- and β-mobility CRP preparations were obtained following purification on DEAE or Sephadex G 200 filtration chromatography. A highly satisfactory anti-CRP antiserum was raised in rabbits with lecithin prepared CRP from liver tissue.     

Microparticle-enhanced nephelometric immunoassay for human C-reactive protein.

Polyfunctional hydrophilic microspheres of 125-nm diameter can be produced by copolymerization of acrylic monomers. Purified c-reactive protein (CRP) was covalently bound to these new micropheres, and the conjugate obtained was used as reagent in a microparticle-enhanced nephelometric immunoassay for human CRP. This assay was based on the measure, with a specially designed nephelometer, of the light scattered by aggregates formed during the immunoagglutination of the conjugate with anti-CRP antiserum. Sensitive inhibition of this agglutination by free CRP (6 ng/ml) allowed CRP quantitation in highly diluted serum samples (1/500-1/2,000), excluding any interference or sample pretreatment. The CRP assay was easy to perform (no washing or phase separation), reliable (coefficients of variation ranged from 1.3% to 9.3% for within-run and between-run determinations), and accurate (mean percentage of recovery: 104%; correlation coefficients with accepted analytical methods greater than or equal to 0.97) over a large range of concentrations. The inhibition mode excluded errors in the antigen excess zone and provided total security at high concentrations.     

Lymphocytes binding C-reactive protein during acute rheumatic fever.

Lymphocytes binding C-reactive protein (CRP) were studied in 31 patients with acute rheumatic fever and 30 controls who were children. Marked elevations in both proportions and absolute numbers of CRP-binding lymphocytes were recorded in rheumatic fever (P less than 0.001). No clear correlation was noted between plasma CRP as quantitated by radioimmunoassay and proportions or numbers of CRP-binding cells. Double-labeling experiments indicated that 60-80% of CRP-binding lymphocytes also showed Fc receptors reacting with fluorescein-conjugated IgG aggregates. Passage of lymphocytes over Ig--anti-IgG columns, removed cells bearing surface Ig but not CRP-binding lymphocytes. Studies of T-cell subpopulations indicated no overlap between Tmicron- and CRP-binding cells; however about half of Tgamma-cells showed concurrent CRP binding. "Active" T-cell rosetting cells did not bind CRP. A 12-15-h incubation of lymphocytes at 37 degrees C in 5% CO2-air showed persistence of CRP binding in substantial proportions of cells particularly in acute rheumatic fever. CRP-binding lymphocytes may represent a marker for immunologically committed cells in acute rheumatic fever.     

Circulating immune complexes and immunoglobulin A rheumatoid factor in patients with mesangial immunoglobulin A nephropathies.

Circulating immune complexes (CIC) containing IgA and C3 were elevated in 48% of IgA nephropathy patients; IgA1 was the predominant subclass. IgA1-IgG CIC were detected in 44%, IgA2-IgG CIC in 7%, and IgM-IgA1 CIC in 16% of the patients. No IgM-IgA2 CIC were detectable. Sucrose gradient ultracentrifugation indicated that IgG-IgA1 CIC were predominantly of intermediate (13-19S) size whereas IgA1-C3 CIC sedimented from 11S to 19S. At acid pH, isolated CIC revealed the presence of substantial amounts of 7S IgA. One third of the patients had elevated serum IgA rheumatoid factor (RF) of both polymeric and monomeric forms despite normal levels of IgM-RF; 87% of patients with elevated IgA-RF had IgA1-IgG CIC. These results indicate that the IgA1 component of CIC in patients with IgA nephropathy is not necessarily of mucosal origin and suggest that a portion of these CIC consists of IgA RF immunologically complexed with autologous IgG.     

Localization of a single binding site for immunoglobulin light chains on human Tamm-Horsfall glycoprotein.

Cast nephropathy is a severe complication of multiple myeloma. Binding of filtered monoclonal light chains (LC) with Tamm-Horsfall glycoprotein (THP) triggers heterotypic aggregation of these two proteins to form casts in the distal nephron of the kidney. To localize the LC binding site on THP, human THP was deglycosylated and underwent limited trypsin digestion in the presence or absence of a nephrotoxic LC known to bind THP. A 29.6-kD band was protected from trypsin digestion by the addition of LC. NH2-terminal amino acid sequence and amino acid analyses revealed this band was located between the 6th and 287th amino acid residues of THP. Six peptides located within this 29.6-kD fragment were synthesized and used as potential inhibitors of binding or aggregation of five different nephrotoxic LCs with THP. Peptide AHWSGHCCL (from amino acid 225 to 233) completely inhibited binding and aggregation of these proteins. Optimal inhibition required a cystine residue in this peptide. Truncation experiments demonstrated the entire sequence was necessary for ideal inhibition and the histidine residue explained the effects of pH on binding. These studies provided a basis for further study of LC-THP interaction and a potential approach toward the prevention of cast nephropathy.     

C-reactive protein and platelet activating factor complexes induce production and release of interleukin-1 by human monocytes

Stimulation of interleukin-1 (IL-1) in peripheral blood monocytes exposed to C-reactive protein (CRP) and C-reactive protein-platelet-activating factor (CRP-PAF) complexes was demonstrated in vitro in this study. Significant synthesis of intracellular IL-1, 2-5-fold increases in the number of adherent monocytes as determined by indirect immunofluorescence using rabbit anti-IL-1 and goat antirabbit IgG-fluorescein isothiocynate, was shown. Both CRP alone (10 μg/ml) and especially CRP-PAF mixture (10 μg CRP/1 ug PAF/ml) showed maximum stimulation of IL-1 of 41 and 95 cells/10 field examined, respectively. CRP alone examined in a dose-response study showed optimum stimulation of IL-1 production at the 1 μg/ml concentration with 57% of monocytes showing fluorescence. CRP at varying concentrations with a constant amount of PAF (1 μg/ml) showed optimum IL-1 staining monocytes (63%) with CRP at 10 μg/ml (10 pg CRP/l μg PAF). Similar significant enhanced extracellular IL-1 production by monocytes exposed to CRP and CRP/PAF mixtures was shown by the mouse thymocyte assay. Supernatants of monocytes exposed to CRP (10 μg/ml) or CRP-PAF (10 μg/l μg/ml) demonstrated mouse IL-1 synthesis of 92 and 110 stimulation indices in the thymocyte assay, respectively. It is suggested that CRP and especially CRP-PAF complexes activate macrophage through their appropriate receptors to production of monokines which modulate the immune system. Thus, CRP appears to function nonspecifically in a behavior reminiscent of specific immunoglobulins.     

Acute lung injury in rat caused by immunoglobulin A immune complexes.

Mouse IgG and IgA, with reactivity to dinitrophenol conjugated to carrier protein, have been isolated from myeloma proteins by means of a variety of affinity techniques. The IgA was predominantly in the dimeric form. The in vitro and in vivo biological activities of IgA-containing immune complexes were assessed in the rat. IgA-containing immune complexes were demonstrated, in a dose-dependent manner in vitro, to activate neutrophils and to generate O.-2. In addition, these immune complexes showed evidence of complement activation in vitro, by the use of immunofixation techniques. When IgA was instilled into the airways of rats and antigen was injected intravenously, acute lung injury occurred, as reflected by increases in lung permeability and morphological changes consisting of blebbing of endothelial cells, intra-alveolar hemorrhage, and fibrin deposition. The lung changes were directly proportional to the amount of IgA instilled into the airways and failed to occur if intravenous injection of antigen was omitted. Lung injury did not occur in animals that received an intravenous injection of antigen in the absence of an airway injection of IgA. Lung injury related to IgA-containing immune complexes was complement dependent but neutrophil independent. In companion studies with mouse IgG-containing immune complexes, acute lung injury also occurred and had morphological features similar to those associated with IgA-induced lung injury except that, in the case of IgG immune complex-induced damage, neutrophils were more evident. Acute lung injury induced by IgG-containing immune complexes, whether of mouse or rabbit origin, was complement and neutrophil dependent. The similarities and differences between IgG- and IgA-associated acute immune complex-induced injury of rat lung were reinforced by the use of morphometry techniques. Studies with another monoclonal IgA antibody-containing antigen-binding activity to phosphorylcholine also demonstrated the ability of IgA antibody to cause acute lung injury in the rat. Neither antibody alone nor antigen (phosphorylcholine linked to bovine serum albumin) alone produced evidence of lung injury. These studies indicate for the first time that immune complexes containing IgA have lung-damaging properties and that the pathogenic mechanisms are different from those associated with IgG-associated immune complex-induced acute lung injury.     

C-reactive protein and high-sensitivity C-reactive protein: an update for clinicians

The measurement of C-reactive protein (CRP) using both standard and high-sensitivity CRP (hs-CRP) assays is becoming common in clinical practice. This article addresses the causes of CRP elevation and the use of different CRP assays in internal medicine, including cardiology, gastroenterology, rheumatology, infectious diseases, and oncology. We focus on the recent medical literature on the use of hs-CRP in cardiovascular disease risk stratification and management, including updated screening guidelines on the use of hs-CRP, such as those issued in 2009 by the Canadian Cardiovascular Society. We also discuss the Reynolds Risk Score, which incorporates hs-CRP and family history with more standard cardiovascular risk factors (eg, tobacco use, hypertension, and dyslipidemia) and frequently leads to improved recategorization of cardiovascular disease risk levels. As the recently completed Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER) trial indicated that statin therapy decreases the vascular events among persons with elevated hs-CRP by half, even when cholesterol levels are low, the inclusion of information on hs-CRP values with other cardiovascular risk factors may assist physicians in medical decision making for patients.     

Development of an ELISA method for detecting immune complexes between tissue-nonspecific alkaline phosphatase and immunoglobulin G

A convenient method for measuring immune complexes between tissue-nonspecific alkaline phosphatase (TNSALP) and immunoglobulin G (IgG) (i.e., TNSALP-IgG) would be highly useful for routine analyses. Here, we identified a surface-active agent that would dissolve membrane but not dissociate TNSALP-IgG complexes. Next, we developed an enzyme-linked immunosorbent assay (ELISA) method for detecting TNSALP-IgG complexes with two monoclonal antibodies (MoAbs): 3-29-3R was coated on assay plates and captured TNSALP-IgG from a specimen; an horseradish peroxidase (HRP)-conjugated anti-human IgG1 then reacted with captured TNSALP-IgG to form an "immunocomplex sandwich." The immunocomplex was detected via the absorbance of an HRP substrate, resulting in a semiquantitative assay. The mean absorbance of 0.195 (n=5) measured in sera from healthy donors was designated as an arbitrary unit (AU/mL) of TNSALP-IgG concentration. The ELISA values of patient sera known to contain TNSALP-IgG complexes were greater than those of normal sera (normal, 1.86 plusmn; 0.61; patient, 9.30 plusmn; 5.44), and these data were confirmed by electrophoresis. Thus, the ELISA could detect TNSALP-IgG complexes. The intraassay coefficient of variation (CV) was within 7.4% and analytical recovery was excellent. There was no significant interference from hemolytic, lipemic, or icteric serum. In summary, an ELISA using 3-29-3R MoAb and HRP-conjugated anti-human IgG1 constitutes a reliable and convenient method for the semiquantitative detection of TNSALP-IgG complexes in human serum.     

Use of solid-phase Clq-ELISA for detecting circulating immune complexes in human diseases

The authors provide the results of studying the methodologic characteristics and of the clinical trial of the C1q-ELISA for detection of the circulating immune complexes. The C1q-ELISA is shown to have a high sensitivity and to make it possible to identify 3 micrograms/ml and more of aggregated gamma-globulin. The method can use for detection of CIC E (ab')2-fragments of antibodies to IgG of man and protein A labeled with peroxidase. Using the method, the circulating immune complexes were identified in the sera of 50% of patients with systemic lupus erythematosus, of 30% of patients with dilated cardiomyopathy and of 53% of patients with nonspecific aortoarteritis. It is concluded that the C1q-ELISA can be used in clinical practice for detecting the circulating immune complexes in different diseases of man.