Richard F. Mortensen

C-reactive protein, inflammation, and innate immunity

The circulating acute phase reactant C-reactive protein (CRP) has traditionally been characterized as an effector of nonclonal host resistance since it activates the classical complement cascade and mediates phagocytosis, but it is also capable of regulating inflammation. The three-dimensional structure of human CRP has revealed the molecular basis for complement activation and binding of phosphate monoesters. CRP gene expression by liver hepatocytes in response to cytokines (IL-1β and IL-6) released in tissues requires several transcription factors which interact. Elevated levels of CRP are a prognostic marker for coronary artery disease; however, the role of CRP in atheriosclerosis remains unknown. CRP also mediates direct host protection to some microbial pathogens via its opsonic activity through certain Fcγ-receptors. The CRP response may be one of the links between nonspecific innate immunity and specific clonal immunity.

Regulation of phagocytic leukocyte activities by C-reactive protein.

The classic acute phase reactant C‐reactive protein (CRP) is classified as an effector of innate host resistance because it activates the classical complement cascade and is opsonic. The latter action occurs via specific CRP receptors (CRP‐R) that have recently been identified as both FcγRI and FcγRII on human phagocytic leukocytes. New findings also suggest an anti‐inflammatory role for CRP because it modulates endotoxin shock and inhibits chemotaxis and the respiratory burst of neutrophils. CRP inhibited phorbol myristate acetate‐induced superoxide (O) production more efficiently than the fMLP‐triggered response. An examination of the inhibition of the protein kinase C (PKC)‐dependent assembly of the NADPH oxidase complex revealed that both phosphorylation and translocation of PKC‐β2 to the membrane were inhibited by a threshold acute phase dose of ∼50 μg/mL CRP. Translocation to the membrane and serine‐phosphorylation of the major cytosolic p47‐phox component of the NADPH oxidase complex was inhibited by CRP. CRP also inhibited membrane localization of activated Rac2, the small G protein regulator of the assembly of the oxidase components in activated neutrophils as well as the cytoskeleton during chemotactic movement. CRP‐mediated regulation occurs via the CRP‐R because an IgM mouse mAb to the human CRP‐R mimicked CRP‐induced inhibition of O production and chemotaxis. CRP may serve as an antiinflammatory regulator of activities at sites of tissue damage where it selectively accumulates and thus influences neutrophil infiltration and polymorphonuclear neutrophil activities. By contrast, CRP activates cells of the monocyte/macrophage lineage, suggesting differential regulation of these two leukocyte populations at the level of signaling. CRP appears to be a multifunctional protein with the capability of exerting both effector functions for innate host resistance, as well as exerting specific anti‐inflammatory effects. J. Leukoc. Biol. 67: 495–500; 2000.

C-Reactive Protein Induces Signaling Through FcγRIIa on HL-60 Granulocytes

Human C-reactive protein (CRP) at acute phase levels of 10–200 μg/ml triggered the phosphorylation of FcγRIIa, Syk kinase, and phospholipase Cγ2 in granulocytic HL-60 cells. CRP also stimulated translocation to the membrane of both phospholipase Cγ2 and phosphatidylinositol-3-kinase. The signaling response triggered by CRP was a rapid, early event with kinetics similar to the response elicited by human IgG. Both soluble-aggregated CRP and monomeric CRP cross-linked FcγRII to generate a signal of the same intensity. The results are consistent with signaling through the intrinsic immunoreceptor tyrosine-based activation motif of the cytoplasmic domain of FcγRIIa, the major CRP-receptor on monocytes and neutrophils that is responsible for CRP-mediated phagocytosis. The signaling events driven by CRP have the potential to regulate infiltrating neutrophil activities.

Receptor-mediated binding of the acute-phase reactant mouse serum amyloid P-component (SAP) to macrophages

Serum amyloid P-component (SAP) is a major acute phase protein of mice which we have previously shown increases the bactericidal activity of elicited, inflammatory macrophages (M phi). The presence of specific receptors for mouse SAP on M phi was demonstrated and the receptor-ligand (SAP) interaction characterized. Purified 125I-labeled mouse SAP binds to elicited M phi with the characteristics of a receptor-mediated event, i.e., the binding was saturable, specific, and reversible. A single type of receptor population was detected with an affinity of 5 x 10(-8) M (KD) and the calculated number of receptor sites per cell was approximately 10(5). Binding of SAP to M phi required Ca2+ or Mg2+ and was inhibited at a pH less than or equal to 5.6. Activated M phi from mice given BCG bind less SAP than nonactivated M phi. Activation of M phi with mouse interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) also decreased their SAP binding capacity. SAP is a glycosylated protein with a high mannose content; therefore mannose and other sugars were tested for inhibition of binding. Specific binding of SAP was inhibited by less than 1 mM concentrations of mannose 6-P, mannose 1-P, and mannose; however, other monosaccharides did not inhibit the binding. Removal of the oligosaccharide from SAP with an endoglycosidase specific for N-linked carbohydrate reduced the binding of SAP to M phi. The pattern of inhibition by sugars, the divalent cation requirement, and the sensitivity to low pH indicate that the receptor binding SAP is the cation-dependent mannose 6-P receptor, or a closely related receptor. The results suggest that SAP may alter or trigger M phi functions associated with inflammation by binding to glycoprotein receptors.

Binding of human C-reactive protein (CRP) to plasma fibronectin occurs via the phosphorylcholine-binding site☆

Human C-reactive protein (CRP) is an acute phase reactant that is selectively deposited at sites of tissue damage. CRP binds with high affinity to purified plasma fibronectin (Fn) when the Fn is immobilized on a surface or matrix via either specific IgG antibody or by gelatin. The CRP to Fn binding is saturable at a molar ratio of CRP/Fn of approximately 9 with a Kd = 1.47 x 10(-7) M and requires Ca2+. The binding site on Fn for CRP was localized to the C-terminal portion by using monoclonal antibodies (mAbs) to Fn as competitive inhibitors of CRP. The binding involves the phosphorylcholine (PC)-binding site of CRP since the addition of PC inhibits binding to Fn and those mAbs to CRP that bind at or near the PC-binding site selectively inhibit the CRP to Fn binding. In addition the mouse IgA myeloma protein TEPC-15, which is specific for PC, also competes with CRP for binding sites on Fn. A mAb to the mouse PC-binding idiotype T-15, which also reacts with the PC-binding site of CRP, inhibits the binding of CRP to Fn. The findings suggest that CRP may play a role in the formation of the extracellular matrix needed for tissue repair. The CRP-Fn interaction may be one of the explanations for the observation of selective deposition of CRP at sites of tissue injury.

In vitro induction of hepatocyte synthesis of the acute phase reactant mouse serum amyloid P-component by macrophages and IL 1.

The in vitro culture conditions for the induction and synthesis of the mouse acute phase reactant, serum amyloid P‐component (SAP), were established using isolated hepatocytes. SAP synthesis was five to eight times greater with hepatocytes isolated from mice during the acute phase of inflammation vs. hepatocytes obtained from untreated mice. The induction of SAP synthesis in normal hepatocytes for LPS‐unresponsive mice was macrophage dependent. Activated macrophages provided the most “helper” activity for SAP production. Partially purified mouse IL 1 from the P388D1 macrophage line also induced SAP synthesis. Only four IL 1 units/ml were required for optimal SAP induction. The addition of IL 1 in the presence of elicited macrophages provided an additive effect on hepatocyte SAP synthesis. The SAP‐inducing activity of IL 1 copurified with its thymocyte‐stimulating activity and was associated with a 11 to 25‐Kd MW polypeptide. Phenylglyoxal treatment of IL 1 inactivated its thymocyte stimulating activity but not its SAP inducing potential. Inhibition of m‐RNA synthesis, protein synthesis, N‐glycosylation, and protein secretion effectively prevented in vitro hepatocyte SAP production.

Internalization and degradation of receptor bound C-reactive protein by U-937 cells : induction of H2O2 production and tumoricidal activity

The presence of a membrane receptor for C-reactive protein (CRP-R) on the human monocytic cell line U-937 was the basis for determining the metabolic fate of the receptor-bound ligand and the functional response of the cells to CRP. Internalized [125I]CRP was measured by removing cell surface-bound [125I]CRP with pronase. Warming cells to 37 degrees C resulted in the internalization of approx. 50% of the receptor-bound [125I]CRP or receptor-bound [125I]CRP-PC-KLH complexes. U-937 cells degraded about 25% of the internalized [125I]CRP into TCA-soluble radiolabeled products. The lysosomotrophic agents (chloroquine, NH4Cl) greatly decreased the extent of CRP degradation without altering binding or internalization. In addition, a pH less than 4.0 resulted in dissociation of receptor-bound [125I]CRP. Treatment of U-937 cell with monensin, a carboxylic ionophore which prevents receptor recycling, resulted in accumulation of internalized [125I]CRP. Therefore, it appears that the CRP-R complex is internalized into an endosomal compartment where the CRP is uncoupled from its receptor and subsequently degraded. CRP initiated the differentiation of the U-937 cells so that they acquired the ability to produce H2O2 and also display in vitro tumoricidal activity. The results support the concept that internalization and degradation of CRP leads to the activation of monocytes during inflammation.

IL-1 and IL-6 mediate increased production and synthesis by hepatocytes of acute-phase reactant mouse serum amyloid P-component (SAP)

Primary mouse hepatocytes exposed to the inflammatory cytokines IL-1 and IL-6 in vitro displayed an increase in the production of the major acute-phase reactant, serum amyloid P-component (SAP). Antiserum to recombinant human IL-6 selectively neutralized the SAP-inducing activity secreted by human diploid fibroblasts. Purified mouse interferon-Β-(IFN-Β), but not IFN-α, also induced SAP production. Addition of 0.05 ng/ml of recombinant mouse IL-1α induced a 10-fold increase in SAP production, whereas recombinant human and recombinant mouse IL-6 displayed optimal SAP-inducing activity of four-fold and seven-fold at 10 ng/ ml and 1 unit/ml/2×105 mouse hepatocytes, respectively. The SAP-inducing activity was neutralized by antibodies to each of the recombinant cytokines. The kinetics of the SAP response in vitro was similar for all of the cytokines. Addition of a mixture of IL-1 and IL-6 to the hepatocytes resulted in SAP production that was not synergistic, but additive, over a range of concentrations for each cytokine. The increase in SAP production mediated by the cytokines was in part the result of an increase in the level of SAP mRNA. Metabolic incorporation of [35S]methionine into mouse SAP occurred in response to both IL-1 and IL-6. Therefore, mouse SAP should be classified among the subset of acute-phase proteins that can be induced by the direct action of either IL-1 or IL-6 on hepatocytes.

Mouse serum amyloid P-component (SAP) levels controlled by a locus on chromosome 1.

Recombinant inbred strains were used to demonstrate the existence of a major locus on chromosome 1, designated Sap, which controls the endogenous concentration of the mouse acute phase reactant, serum amyloid P-component (SAP). Levels of SAP were associated with alleles at the Ly-9 locus in two sets of RI strains: BXD (C57BL/6J × DBA/2) and BXH (C57BL/6J × C3H/HeJ). Low endogenous levels of SAP were present in the C57BL/6J progenitor strain and in most of the RI strains which inherited the Ly-9ballele. High levels of SAP were present in the DBA/2J and C3H/HeJ progenitors and in most of the RI strains which inherited the Ly-9aallele. In the BXD strains 91% of the genetic variation of SAP levels was accounted for by segregation at the Ly-9 locus while an additional 9% was attributed to genetic factors unlinked to Ly-9. In the BXH strains the percentage of genetic variation accounted for by Ly-9 segregation was reduced to 46%, while 54% was accounted for by other genetic factors. Because of background genetic variation it was not possible to detect any crossovers between Sap and Ly-9. However, in the BXD strains the linkage between Sap and Ly-9 appears to be quite close. The B6.C-H-25ccongenic strain, which carries a segment of BALB/c chromosome 1 including the minor histocompatibility locus H-25 on a C57BL/6By background, had the same endogenous SAP level as the BALB/c donor strain.

Enhanced interleukin 1 (IL-1) production mediated by mouse serum amyloid P component

Purified serum amyloid P component (SAP), the major acute-phase reactant of mice, induces enhanced interleukin 1 (IL-1) production by elicited monocytes/macrophages in vitro. SAP also enhanced IL-1 elaboration by macrophages from lipopolysaccharide (LPS)-low responder mice and in the presence of polymyxin B, indicating that the small amounts of LPS present in the SAP preparation did not augment IL-1 production. Concentrations of SAP of 0.1 to 10.0 micrograms/ml enhanced IL-1 production by elicited and bacillus Calmette-Guerin (BCG)-activated peritoneal macrophages, but not by resident peritoneal macrophages. The inflammation-induced monocyte/macrophage population displayed selective binding of SAP. The mouse macrophage line P388D1, also could bind SAP and display enhanced IL-1 production in response to SAP. SAP did not bind to the macrophage cell line RAW264.7 nor did it enhance IL-1 secretion by this line. The results suggest that this acute-phase reactant has the potential to enhance inflammatory and immunological events mediated by IL-1.