Characterization and isolation of a C-reactive protein receptor from the human monocytic cell line U-937

Human C-reactive protein (CRP) is an acute phase reactant that is opsonic and an activator of macrophage tumoricidal function. CRP also activates the classical C cascade. These activities suggest that CRP might interact with monocytes/macrophages via specific receptors in a manner analogous to the interaction of IgG with FcR. With the use of radio-labeled human CRP, we have observed specific binding of CRP to human blood monocytes and the human monocytic cell line U-937. Binding was saturable at a pathophysiologic concentration of CRP, with an estimated KD of 9.5 x 10(-8) M and 3.6 x 10(5) binding sites/cell. Specific binding was inhibited by polyclonal human IgG as well as an IgG1 myeloma. In the converse experiment, CRP failed to inhibit specific [125I]IgG binding. The mAb IV.3, which inhibits binding of IgG immune complexes to FcRII, did not inhibit CRP binding. A 100-fold excess of phosphorylcholine or the phosphorylcholine binding peptide of CRP (residues 47-63) failed to inhibit binding. Although human rIFN-gamma and PMA increased FcRI expression, these reagents had no affect on CRP receptor expression. A single membrane protein of 38 to 41 kDa from U-937 cells was chemically cross-linked to [125I]CRP; the cross-linking was inhibited by human IgG1 but not the IV.3 mAb. Furthermore, two membrane proteins with a Mr of 38 to 40 kDa and 58 to 60 kDa were isolated by CRP ligand-affinity chromatography. These proteins were of a distinct size from those isolated for FcRI from an IgG ligand matrix. These studies demonstrate specific binding of human CRP to a human monocytic cell line via receptors that are distinct from the IgG FcR and implicate CRP in nonspecific, preimmune host defense reaction mediated by cells of the monocytic lineage.     

Biosynthesis of human C-reactive protein in cultured hepatoma cells is induced by a monocyte factor(s) other than interleukin-1

An in vitro cell culture system utilizing continuous human liver cells has been developed which, upon specific induction, will respond by synthesizing, de novo, the prototype acute phase reactant, C-reactive protein (CRP). Induction of CRP in vitro is not brought about by the types of hormones, steroids, and chemicals which affect other acute phase proteins. In particular, interleukin-1 thought to be directly responsible for acute phase induction is not found to be active. Direct testing of other purified biological response modifiers, i.e. alpha, beta, and gamma-interferon, interleukin-2, and tumor necrosis factor, demonstrates no inducing activity. However, we find that human peripheral blood monocytes, stimulated by endotoxin, produce a factor(s) which directly induces CRP synthesis in hepatoma cells. In addition, the human promyelocyte-like cell line HL-60 in the presence of phorbol ester and certain T-cell lines containing human retroviruses also produce this CRP-inducing factor(s). Isolation and partial purification of the CRP-inducing factor(s) indicate that it is a protein(s) with a molecular weight of approximately 30,000.     

Induction of inflammatory cytokine release from cultured human monocytes by C-reactive protein.

The human acute phase protein, C-reactive protein (CRP), is capable of specifically binding to and modulating the function of mononuclear phagocytes. To investigate whether CRP can also affect the capacity of these cells to produce inflammatory cytokines, enzyme immunoassays and Western blot techniques were used to quantitate interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) produced by freshly-isolated normal human monocytes. CRP induced the rapid release of each cytokine, with significantly elevated levels in culture supernatants at 4 hours and maximal levels of TNF-alpha at 8 hours, and of IL-1 beta and IL-6 at 16 hours of culture. The effects of CRP were dose-dependent; greater than 10-fold increases of each cytokine were observed following culture with greater than or equal to 50 micrograms/ml CRP, concentrations which are often found in the presence of moderate to severe inflammation or tissue injury. The induction of cytokine release by CRP was unaffected by inclusion of 25 micrograms/ml polymyxin-B in culture media, but was completely abrogated by prior boiling of the CRP, a procedure which had no effect on induction of monocyte cytokine release by lipopolysaccharide. The dose-dependent induction of inflammatory cytokines by CRP provides further support for the hypothesis that interaction with mononuclear phagocytes constitutes an important biological role for this acute phase protein.     

Inhibition of the polyclonal antibody plaque-forming cell response of human B lymphocytes by C-reactive protein (CRP) and CRP complexes

The pokeweed mitogen (PWM)-induced generation of polyclonal immunoglobulin-secreting cells (ISC), as measured by reverse hemolytic plaque formation of protein A-coated sheep E by human blood mononuclear cells, was inhibited by both purified human C-reactive protein (CRP) and CRP-C-polysaccharide (CRP-CPS) complexes. CRP and CRP-CPS mediated the suppression by binding and activating monocytes and T cells with IgG Fc receptors. The extent of suppression was dependent on CRP concentration and the CRP/CPS ratio and was similar to that obtained with IgG immune complexes. In contrast, CRP did not alter the number of ISC formed in response to the relatively T-independent polyclonal activator, protein A-bearing staphylococci. Suppression of ISC formation was most likely confined to events associated with the terminal stages of B-cell differentiation since no effect of CRP or CRP-CPS on the blastogenic response to polyclonal B-cell activators (PBA) was detected. These findings indicate that the acute phase reactant CRP has the potential to modulate antibody responses during the course of an inflammatory response.     

Heterogeneous nature of the acute phase response. Differential regulation of human serum amyloid A, C-reactive protein, and other acute phase proteins by cytokines in Hep 3B cells

Because a number of different cytokines have been reported to regulate the synthesis of human, murine, and rat acute phase proteins (APP), we studied the effect of cytokines on production of several major human APP in a single system, the human hepatoma cell line Hep 3B. Conditioned medium (CM) prepared from human blood monocytes activated with LPS in the presence of dexamethasone led to substantial induction of serum amyloid A (SAA) and C-reactive protein (CRP) synthesis whereas the defined cytokines IL-1 beta, TNF alpha, and medium from a human keratinocyte cell line (COLO-16), containing hepatocyte-stimulating factor activity, failed to induce these two major APP. Induction of SAA and CRP was accompanied by an increase in concentration of their specific mRNA. Size fractionation of CM from activated monocytes by fast protein liquid chromatography indicated that SAA- and CRP-inducing activity eluted as a single peak with a Mr of approximately 18 kDa. alpha 1-Antitrypsin, which also failed to respond to IL-1 beta or TNF alpha, was induced by both CM and medium from COLO-16 cells. The induction of AT by CM was accompanied by an increase in specific mRNA. Induction of ceruloplasmin and alpha 1-antichymotrypsin and decrease in the synthesis of albumin was achieved by both CM and IL-1 beta. Ceruloplasmin and albumin responded in a comparable fashion to both TNF alpha and medium from COLO-16 cells; the response of ACT to these cytokines was not evaluated. These results indicate that human SAA and CRP are induced in Hep 3B cells by products of activated monocytes but not by IL-1 beta, TNF-alpha, or some hepatocyte-stimulating factor preparations and that a group of heterogeneous mechanisms are involved in the induction of the various human APP.     

Experimental allergic encephalomyelitis is inhibited in transgenic mice expressing human C-reactive protein

We show here using a transgenic model that human C-reactive protein (CRP) protects against experimental allergic encephalomyelitis (EAE) in C57BL/6 mice. In transgenic compared with wild-type females, the duration of the human CRP acute phase response that accompanies the inductive phase of active EAE correlates with a delay in disease onset. In transgenic males, which have higher human CRP expression than females do, EAE is delayed, and its severity is reduced relative to same-sex controls. Furthermore, in male transgenics, there is little or no infiltration of the spinal cord by CD3(+) T cells and CD11b(+) monocytes and macrophages, and EAE is sometimes prevented altogether. CRP transgenics also resist EAE induced passively by transfer of encephalitogenic T cells from wild-type donors. Human CRP has three effects on cultured encephalitogenic cells that could contribute to the protective effect observed in vivo: 1) CRP inhibits encephalitogenic peptide-induced proliferation of T cells; 2) CRP inhibits production of inflammatory cytokines (TNF-alpha, IFN-gamma) and chemokines (macrophage-inflammatory protein-1alpha, RANTES, monocyte chemoattractant protein-1); and 3) CRP increases IL-10 production. All three of these actions are realized in vitro only in the presence of high concentrations of human CRP. The combined data suggest that during the acute phase of inflammation accompanying EAE, the high level of circulating human CRP that is achieved in CRP-transgenic mice inhibits the damaging action of inflammatory cells and/or T cells that otherwise support onset and development of EAE.     

Analysis of the binding of fluorescent C5a and C3a to human peripheral blood leukocytes

Fluorescein-labeled human C5a and C3a were prepared and utilized to analyze the binding of C5a and C3a to human neutrophils and mononuclear cells. The fluorescein derivatives of C5a (Fl-C5a) and C3a (Fl-C3a) contained approximately one fluorescein molecule per molecule of protein. Fl-C5a retained biologic activity as determined by neutrophil O2- production, enzyme release, receptor binding, and reaction with rabbit anti-C5a antibody. Fl-C3a was biologically active as measured by contraction of guinea pig ileal strips, and maintained 87% of its antigenic character when reacted with rabbit anti-human C3a. The binding of Fl-C5a and Fl-C3a to human neutrophils and mononuclear cells was assessed with the use of flow cytometry. Fl-C5a bound to greater than 90% of neutrophils, with an average ED50 ranging from 2.8 to 6.8 nM, depending on the method of analysis. Fl-C5a binding to neutrophils was specific and was not inhibited by the presence of formyl-methionyl-leucyl-phenylalanine (f-MLP), C3a, or casein. Fl-C5a binding was totally blocked by an excess of C5a. C5a des arg partially inhibited the binding of Fl-C5a to neutrophils, but was 1000-fold less effective than C5a. Similar experiments with mononuclear cells showed that Fl-C5a was bound by monocytes but not by lymphocytes. Fl-C5a binding to monocytes was blocked totally by C5a but not by C3a or f-MLP. Comparative binding studies with neutrophils, monocytes, and lymphocytes showed that Fl-C5a was bound by an average of 93% +/- 4 of neutrophils, 68% +/- 9 of monocytes, and 6% +/- 3 of lymphocytes. Fl-C3a did not show significant binding to neutrophils, monocytes, or lymphocytes. These studies demonstrate that fluorescein derivatives of C5a and C3a can be prepared with retention of biologic activity, and provide a means to evaluate the binding of C5a to individual cells.     

Lectin specificity and binding characteristics of human C-reactive protein.

C-reactive protein (CRP) is thought to play an important role in immunomodulation. The exact biologic function of this pentraxin protein is, however, still unclear. Here we report experiments designed to further characterize the binding properties of CRP. Using purified human CRP it could be shown that CRP immobilized onto polystyrene surfaces or onto latex beads binds distinct plasma glycoproteins including IgG, asialofetuin, asialo-beta 2-glycoprotein I and, likewise, synthetic glycoproteins as a lectin, exhibiting binding specificity for terminal galactosyl residues of the glycoprotein glycans. Binding of CRP to IgA, IgM, IgG, asialofetuin, asialo-beta 2-glycoprotein I and to synthetic glycoproteins requires immobilization onto surfaces of both CRP and the ligand. Fibronectin and fibrinogen are bound by surface-immobilized CRP also in soluble phase. Comparing various mono-, di-, and trisaccharides as competitive inhibitors of the lectin binding activity of CRP, only beta-D-Gal-(1-3)-D-GalNAc, beta-D-Gal-(1-4)-D-GalNAc, and beta-D-Gal-(1-4)-beta-D-Gal-(1-4)-D-GlcNAc had significant inhibitory power at a concentration of 8 mmol/liter. Binding activity of CRP was pH-dependent with an optimum at pH 5 to 6 and was reduced by 90% when pH was shifted from 6 to the physiologic pH value of 7.4. CRP exhibited lectin-like properties with binding specificity for galactosyl residues also when bound to K-562 erythroleukemia cells. It is therefore suggested that CRP immobilized onto surfaces exhibits lectin activity toward galactosyl groups preferentially in a mildly acidic environment as present at sites of inflammation.     

A synthetic peptide corresponding to the phosphorylcholine (PC)-binding region of human C-reactive protein possesses the TEPC-15 myeloma PC-idiotype.

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. CRP molecules from all species display Ca2(+)-dependent binding to phosphorylcholine (PC). The conserved PC-binding region of CRP corresponds to amino acids 51-66 within the human CRP sequence. A synthetic peptide composed of residues 47-63 of human CRP was previously shown to possess PC binding activity. The charged amino acids at positions 57, 58, 60, and 62 of this synthetic peptide were critical for PC-binding based on lower binding activity of synthetic peptides containing uncharged residues at these positions. The PC-binding peptide was used to generate mouse mAb that were tested for reactivity with intact CRP and with the TEPC-15 (T-15) mouse myeloma protein that also binds PC. The PC-binding peptide of CRP was recognized by two mAb specific for the T-15 Id. One of the mAb generated against the PC-binding peptide of CRP (IID6.2) recognized an epitope on the T-15 protein that was also recognized by the near-binding site-specific mAb (F6) to the T-15 PC-Id. Binding of IID6.2 to T-15 myeloma protein was not inhibited by PC and did not require Ca2+; however, binding was inhibited by the synthetic PC-binding peptide itself. Recognition of synthetic peptides containing uncharged amino acid substitutions by mAb F6 and IID6.2 was greatly reduced indicating that the shared epitope on T-15 and CRP was composed of similar charged residues. Therefore, CRP displays the same idiotope as an antibody that shares its specificity for the hapten, PC.     

Native and modified C-reactive protein bind different receptors on human neutrophils

Native C-reactive protein (CRP) is a planar pentamer of identical subunits expressed at high serum levels during the acute phase of inflammation. At inflammatory sites, an isomeric form termed modified CRP (mCRP) is expressed and reveals neoantigenic epitopes associated with modified monomeric CRP subunits. mCRP cannot assume the native pentameric conformation but rather forms a nonpentameric aggregate of monomers. While native CRP inhibits neutrophil movement in vitro and in vivo, the effect of mCRP on neutrophil movement has not been reported. To model the biological function and biochemical interaction of mCRP on neutrophils, in vitro chemotaxis and binding experiments were performed using mCRP. Reported here, mCRP effectively inhibited fMLP-induced chemotaxis similar to native CRP. Additionally, mCRP increased binding of labeled native CRP to neutrophils. This increased binding occurred by direct protein-protein interaction of the C-terminus thereby implicating the CRP(199-206) sequence. Binding of mCRP to neutrophils was blocked by anti-CD16 monoclonal antibody whereas native CRP was not. These results suggest that modified forms of CRP inhibit chemotaxis, a function similar to native CRP, but that mCRP and native molecule bind different receptors on human neutrophils.     

Effects of C-reactive protein on human neutrophil granulocytes challenged with N-formyl-methionyl-leucyl-phenylalanine and platelet-activating factor

The effects of C-reactive protein (CRP), the prototypical acute-phase reactant were studied on human polymorphonuclear leukocytes (PMNL) challenged with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF). CRP at 8-64 micrograms/ml concentrations inhibited degranulation and superoxide production by PMNL in time-, and dose-dependent manner and stabilized PMNL membranes against the lytic effect of lysophosphatidylcholine. CRP was also capable of binding PAF and in lesser extent fMLP. Furthermore, CRP, 32 micrograms/ml, diminished specific binding of [3H]-fMLP and [3H]-PAF to PMNL. These findings imply that CRP may play an important protective role during the early phase of acute inflammatory reactions.     

C-reactive protein receptors on the human monocytic cell line U-937. Evidence for additional binding to Fc gamma RI.

C-reactive protein (CRP) is an acute-phase protein that binds to components of damage tissue, activates C, and stimulates phagocytic cells. CRP binding to receptors on monocytic and polymorphonuclear phagocytes has been shown. Recently, CRP-binding proteins of 38 to 40 kDa and 57 to 60 kDa have been identified on the human promonocyte cell line U-937 and the mouse macrophage cell line PU5 1.8, respectively. However, analysis of CRP binding to these cells and to peripheral blood leukocytes suggests that additional CRP receptor sites may be present. Because many studies have shown interactions between CRP binding and IgG binding to leukocytes, we have examined further the CRP binding sites on U-937 cells and determined their relationship to the FcR for IgG (Fc gamma R) expressed on these cells. Our results demonstrate specific saturable binding of CRP to peripheral blood monocytes and U-937 cells, which is readily inhibited by aggregated IgG. Monomeric IgG, which binds specifically to Fc gamma RI, inhibited a maximum of 20% of CRP binding to these cells. mAb 197 and mAb IV.3, which block IgG binding to Fc gamma RI and Fc gamma RII, respectively, failed to inhibit CRP binding to U-937 cells. Two CRP-binding molecules were identified by precipitation of lysates from surface-labeled U-937 cells and cross-linking experiments. One of these had a molecular mass of 43 to 45 kDa, similar to the molecule previously described as the CRPR on U-937 cells. The other had the same mobility by SDS-PAGE as Fc gamma RI. The identity of this protein with Fc gamma RI was confirmed by the ability of both IgG-Sepharose and CRP-Sepharose to preclear the protein from cell lysates and by inhibition of binding to both IgG-Sepharose and CRP-Sepharose by anti-Fc gamma RI mAb 197.     

Regulated export of a secretory protein from the ER of the hepatocyte: a specific binding site retaining C-reactive protein within the ER is downregulated during the acute phase response.

The half-time for secretion of the plasma protein C-reactive protein (CRP) by the hepatocyte decreases markedly in association with its increased synthesis during the acute phase response to tissue injury (Macintyre, S., D. Samols, and I. Kushner. 1985. J. Biol. Chem. 260:4169-4173). In studies in which subcellular fractions were prepared from cells incubated under pulse-chase conditions, CRP was found to be preferentially retained within the ER of normal hepatocytes, but secreted relatively efficiently in cells prepared from rabbits undergoing the acute phase response. On the basis of the detergent-dependency of specific binding of radiolabeled CRP, as well as EM visualization of biotinylated CRP identified with peroxidase-conjugated streptavidin, CRP was found to bind to the lumenal surface of permeabilized rough microsomes, while no binding was detected in Golgi fractions. As judged by both kinetic and equilibrium binding studies, rough microsomes from control rabbits were found to have two classes of specific binding sites for CRP; a high affinity site (Kd = 1 nM, Bmax = 1 pmol CRP/mg microsomal protein) as well as a much lower affinity (Kd = 140 nM) site. In contrast, only the lower affinity class was detected in microsomes isolated from rabbits undergoing the acute phase response. On nitrocellulose blots probed with radiolabeled CRP a 60-kD protein, distinct from BiP, was detected in extracts of rough microsomes isolated from control rabbits, but not in Golgi fractions or rough microsomes from stimulated animals. These findings correlate with previous observations of changes in secretion kinetics of CRP and are consistent with the hypothesis that the intracellular sorting of CRP could be rerouted by downregulation of a specific ER binding site during the acute phase response.     

Heterogeneity in human interleukin-3 receptors. A subclass that binds human granulocyte/macrophage colony stimulating factor

125I-Labeled recombinant human interleukin-3 (IL-3) was used to study the characteristics and distribution of receptors for IL-3 on human cells. Receptors were found on primary monocytes, on some strains of KG-1 cells, and on pre-B cell lines. Binding was rapid at 37 degrees C, while requiring several hours to reach equilibrium at 4 degrees C. Equilibrium binding studies indicated that IL-3 bound to a single class of high affinity receptor (less than 500 receptors/cell) with a Ka of approximately 1 x 10(10) M-1. Inhibition studies revealed that human granulocyte/macrophage colony stimulating factor partially inhibited the binding of 125I-IL-3 to human monocytes but not JM-1 cells. Additional analysis showed that on KG-1 cells, both IL-3 and GM-CSF partially competed specific binding of heterologous radiolabeled ligand, with approximately equivalent capacities. This competition occurred at both 37 and 4 degrees C. These results suggest heterogeneity in the binding sites for IL-3 and GM-CSF in which a subset of receptors binds only IL-3, a subset only GM-CSF, and another subset can bind both, all with high affinity. Additional heterogeneity was suggested by equilibrium binding of 125I-IL-3 to KG-1 cells which revealed a biphasic Scatchard plot containing a low affinity component not observed on monocytes and JM-1 cells.     

Variations in binding characteristics of glycosylated human C-reactive proteins in different pathological conditions

C-reactive protein (CRP) is a clinically important classic acute phase pentameric protein. It is thought to play an important role in immunomodulation. Earlier reports convincingly demonstrated that human CRP is differentially glycosylated in different pathological conditions. Although CRP is considered to be a clinically important molecule, changes in binding characteristics with appropriate ligands with respect to glycosylation remain unexplored. In an effort to demonstrate that these glycosylated molecular variants are capable of modulating their binding activity with different ligands, CRPs were affinity purified from six different clinical samples. Variable amounts of linkage-specific sialic acid derivatives were found in these CRPs with varying tryptophan contents. Differential binding patterns with antibodies against human CRP, human IgG, and other ligands like fibronectin, fetuin, and asialofetuin indicated that the purified CRPs differed significantly in their lectin-like interactions. Thus, we have convincingly demonstrated that differentially induced CRPs exhibited variable binding characteristics. These results may have far reaching practical applications for understanding acute phase responses.     

Effects of C-reactive protein on the lymphoid system. II. Inhibition of mixed lymphocyte reactivity and generation of cytotoxic lymphocytes.

C-reactive protein (CRP), an acute phase reactant which increases in concentration during inflammation, has been found to bind to human T cells and to inhibit certain of their functions. In the present study CRP was found to display a binding specificity for theta-bearing cells from mouse peripheral lymphoid tissue but not for thymus cells. CRP inhibited the proliferative response in a similar manner in both murine and human mixed lymphocyte reactions. This inhibition was prevented by the addition of the CRP substrate, pneumococcal C-polysaccharide (CPS), and was not a result of toxicity of CRP for lymphocytes. By contrast the response of spleen lymphocytes to mitogenic Con A concentrations was not altered by CRP. CRP also exerted an inhibitory effect on the in vitro generation of cytolytic T lymphocytes (CL) in mixed lymphocyte reactions of mouse spleen cells. The expression of the cytolytic process by T cells sensitized either in vivo or in mixed lymphocyte cultures was not altered in the presence of CRP. Therefore, CRP appears to influence the inductive phase of the allograft response and perhaps exerts a regulatory effect on cellular immune responsiveness during inflammatory reactions.     

Characteristics of the binding of human C-reactive protein (CRP) to laminin

Human CRP binds to the basement membrane protein laminin in vitro in a Ca2+-dependent manner via the phosphorylcholine (PC) binding site of C-reactive protein (CRP). The binding was saturable at a molar ratio of 4 (CRP/laminin). The specificity of the binding was shown by inhibition of binding of labeled CRP to laminin by unlabeled CRP, but not by human IgG. Specific binding was optimal in the presence of 5 mM Ca2+, but did not occur in the absence of Ca2+ or in the presence of EDTA. The binding of Ca2+ to CRP causes a conformational change in the molecule, which is required for binding to PC and to laminin. The PC binding site of CRP was implicated in the binding to laminin on the basis of inhibition by both soluble PC and anti-idiotypic mAbs directed to the TEPC-15 PC-binding idiotype found on mouse antibodies to PC. In addition, mouse mAbs specific for the CRP PC binding site displayed decreased reactivity with CRP already bound to laminin. The binding of CRP to laminin provides a possible explanation for selective deposition of CRP at inflamed sites. The CRP-laminin interaction may serve as a means of concentrating CRP at sites of tissue damage so that the CRP might function as a ligand for leukocytes, an event that will result in removal of necrotic tissue and cell debris.     

Monocyte-conditioned medium, interleukin-1, and tumor necrosis factor stimulate the acute phase response in human hepatoma cells in vitro

Human hepatoma cells mimic the acute phase response after treatment with monocyte-conditioned medium. Levels of secreted fibrinogen, alpha-1 acid glycoprotein, C-reactive protein, haptoglobin, and the third component of complement were elevated compared with control levels after 48 h of incubation with conditioned supernatant medium from an enriched fraction of normal peripheral monocytes. Albumin levels declined and alpha-1 antitrypsin remained unchanged. Levels of specific mRNA were measured by hybridization to slot blots and Northern blots and changed in correspondence with protein alterations. Interleukin-1 and tumor necrosis factor stimulated the third component of complement, but did not elevate any other member of the acute phase group and were therefore only partially active in this system. The identification of an in vitro model of the human acute phase response will permit analysis of the molecular basis for coordinate regulation of this group of facultative genes.