Complement Receptors CR1 Research Articles

Soluble CD16/Fc gamma RIII induces maturation of dendritic cells and production of several cytokines including IL-12.

Fc gamma RIII (CD16), a low affinity FcR which binds IgG-containing immune-complexes, exists under membrane-associated forms and under a soluble form (sFc gamma RIII). The latter, present in biological fluids (serum, saliva), is generated by proteolytic cleavage of the two membrane-associated Fc gamma RIII isoforms, Fc gamma RIII-A (expressed by macrophages and NK cells) and Fc gamma RIII-B (expressed exclusively by neutrophils). Herein we demonstrate that dendritic cells (DCs), generated by culturing monocytes with GM-CSF and IL-4, bind biotinylated recombinant sFc gamma RIII. This binding is specific and involves the complement receptor CR3 (CD11b/CD18) and CR4 (CD11c/CD18). Indeed, preincubation of DCs with anti-CD11b and anti-CD11c mAbs decreased by 52% and 62% respectively the binding with sFc gamma RIII. Moreover, electron microscopy showed that binding of gold-labeled sFc gamma RIII to DCs maintained at 4 degrees C occurred within clathrin-coated pits. Once internalized, at 37 degrees C, sFc gamma RIII entered the endocytic pathway and reached the MHC class II compartments. Furthermore, DCs incubated for 48 h with multivalent sFc gamma RIII expressed increased levels of CD40, CD80, CD86, CD54, CD58, HLA class I and class II molecules and decreased levels of CD23 and CD32. These effects result in an increased capacity of DCs to trigger proliferative responses by CD4+ CD45RA+ allogeneic T cells. RT-PCR amplification demonstrated that incubation of DCs for 20 h in the presence of multivalent sFc gamma RIII induced the appearance of GM-CSF and IL-12 p40 mRNA. Among the cytokines constitutively expressed, IL-1 beta and IL-8 were strongly up-regulated whereas IL-6 and IL-12 p35 mRNA were increased to a lesser extent and the expression of MIP-1 alpha mRNA remained constant. Finally, ELISA tests demonstrated that DCs incubated with multivalent sFc gamma RIII secreted the cytokines IL-1 beta, IL-6, IL-8, GM-CSF and IL-12 p75. Thus, while becoming internalized sFc gamma RIII could affect the capacity of DCs to present antigens and, via the induction of accessory molecules and the release of the IL-12 p75 protein, could initiate Th1 type immune response.

Development of a bispecific F(ab')2 conjugate against the complement receptor CR3 of macrophages and a variant CD44 antigen of rat pancreatic adenocarcinoma for redirecting macrophage-mediated tumor cytotoxicity.

A bispecific F(ab')2 antibody conjugate (BAC) was constructed against the complement receptor CR3 of macrophages and variant CD44 (CD44v6) antigen of rat pancreatic adenocarcinoma cells to redirect macrophage-mediated tumor cytotoxicity. The Fab' fragments of monoclonal antibodies (mAb) 1.1ASML and OX42, recognizing the CD44v6 and the CR3 antigens respectively, were chemically coupled at the hinge region using 5,5'-dithiobis(2-nitrobenzoate). The BAC was characterized in vitro for its specific, dual binding capacity to CD44v6 and CR3 antigens. Although the monovalence of the BAC resulted in lower avidities to both the antigens as expected, it was still able to form stable cross-linkages between tumor cells and macrophages in culture leading to the formation of "clump-like" cell aggregates. The in vitro and in vivo tumor-targeting capacity of the BAC was compared with that of the parental antitumor mAb 1.1ASML, which mediates tumor killing by antibody-dependent cell cytotoxicity. These results showed that, even though the bivalent mAb 1.1ASML did not mediate stable cross-linking of target and effector cells, its Fc-receptor-mediated killing of tumor cells was more effective when compared to the BAC. Thus, this study strongly supports the hypothesis that firm persistent binding between effector and target cells per se is not as important as the choice of trigger molecule used for macrophage activation to redirect their tumor cytotoxic potential effectively.

Immunohistochemical demonstration of cellular antigens of the cat defined by anti-human antibodies

A panel of monoclonal antibodies (mAb) and some polyclonal rabbit sera directed against human antigens were studied on cryostat tissue sections of three cats using immunohistochemistry. Reactivity of the antibodies was tested on feline tonsil, intestine, thymus, lymph node and spleen with a three-step immunoperoxidase technique and compared with reactions on human thymus, lymph node and spleen. From a total of 95 antibodies, 28 gave reactivity comparable with that in human tissues. The remaining antibodies gave none or miscellaneous results. The positive reactions in the cat included antibodies directed to adhesion molecules (VLA-2 and VLA-4), to natural killer (NK) cells (CD56, CD57 and NCAM), to complement receptor CR1, to proliferation marker Ki-67 (MIB-1), to endothelial antigens (EN-4, PAL-E and von Willebrand factor) and to structural proteins like vimentin, desmin, collagen type IV and cytokeratin. The identification of these cross-reacting antibodies extends the spectrum of immunological reagents that are now available for the cat, and will thus contribute to the study of the feline immune system.

Soluble Fcgamma receptor type III (FcgammaRIII, CD16) triggers cell activation through interaction with complement receptors.

The type III-B Fcgamma receptor (FcgammaRIII-B) is a glycosyl-phosphatidylinositol-linked receptor found on human neutrophils. A soluble form of FcgammaRIII-B (sCD16) corresponding to the extracellular region of the receptor circulates in plasma. In the present work, we have identified membrane receptors for sCD16. Soluble CD16 bound to CR3 (CDllb/CD18)- and CR4 (CDllc/CD18)- positive leukocytes and cell lines, the labeling was inhibited by anti-CD11b, CD11c or CD18 mAbs, and the up-regulation of CR3 and CR4 led to an increased fixation of sCD16. Transfected eukaryotic cells expressing recombinant CD11b/CD18 or CD11c/CD18 heterodimers but not those expressing CD11a/CD18 bound sCD16. Moreover, the lectin-like binding site of CR3 is probably involved in the interaction with sCD16, as suggested by inhibition studies using mAbs against CR3 or sugars such as N-acetyl D-glucosamine, alpha- or beta-methyl D-glucoside, alpha- or beta-methyl D-mannoside, or zymosan. Thus, the complement receptors CR3 and CR4 are membrane receptors for sCD16. Through this interaction, sCD16 induces a CR3-dependent production of IL-6 and IL-8 by monocytes. These results suggest that sCD16 plays a regulatory role in inflammatory processes and provide a molecular basis for the interaction between FcgammaRIII-B and CR3 described on the cell membrane.

Interleukin-3 Cooperates With Tumor Necrosis Factor α for the Development of Human Dendritic/Langerhans Cells From Cord Blood CD34+ Hematopoietic Progenitor Cells

We have previously shown that tumor necrosis factor (TNF)alpha strongly potentiates the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin (IL)-3-dependent proliferation of CD34+ hematopoietic progenitor cells (HPC) through the recruitment of early progenitors with high proliferative potential. Furthermore, the combination of GM-CSF and TNFalpha allows the generation of large numbers of dendritic/Langerhans cells (D-Lc). Herein, we analyzed whether IL-3, when combined to TNFalpha would, as does GM-CSF, allow the generation of CD1a+ D-Lc. Accordingly, cultures of cord blood CD34+ HPC with IL-3 + TNFalpha yielded 20% to 60% CD14+ cells and 11% to 17% CD1a+ cells, while IL-3 alone did not generate significant numbers of CD1a+ cells. Although the percentage of CD1a+ cells detected in IL3 + TNFalpha was lower than that observed in GM-CSF + TNFalpha (42% to 78%), the strong growth induced by IL-3 + TNFalpha generated as many CD1a+ cells as did GM-CSF + TNFalpha. The CD14+ and CD1a+ cells generated with IL-3 + TNFalpha are similar to CD14+ and CD1a+ cells generated in GM-CSF alone and GM-CSF + TNFalpha, respectively. CD1a+ cells differed from CD14+ cells by (1) dendritic morphology, (2) higher expression of CD1a, CD1c, CD4, CD40, adhesion molecules (CD11c, CD54, CD58), major histocompatibility complex (MHC) class II molecules and CD28 ligands (CD80 and CD86), (3) lack of Fc receptor FcgammaRI (CD64) and complement receptor CR1 (CD35) expression, and (4) stronger induction of allogeneic T-cell proliferation. Thus, in combination with TNFalpha, IL-3 is as potent as GM-CSF for the generation of CD1a+ D-Lc from cord blood CD34+ HPC. The dendritic cell inducing ability of IL-3 may explain why mice with inactivated GM-CSF gene display dendritic cells.

The cellular reactions to experimental intracerebral hemorrhage

The resolution of an intracerebral hemorrhage can be measured by the occurrence of hemosiderin. Extravasation of blood elicits a cellular reaction in the adjacent surviving tissue where the lesion activates resident microglia and attracts many more phagocytes from the blood stream. The signals for this migration into the perifocal reactive zone are not fully understood but it is likely that proteins in the coagulated blood contribute to cellular activation. In order to study the role of plasma proteins in the pathogenesis of the perifocal reactive zone, intracerebral injections of either autologous whole blood (0.1 ml) or an equal volume of washed autologous red blood cells (RBC) in lactated Ringer's solution were made in adult rabbits. The amount of total iron was the same (30 μg). The cellular responses to the injections were studied by iron histochemistry and immunocytochemistry for ferritin, the ferritin repressor protein (FRP), the glial fibrillary acidic protein (GFAP), and the complement receptor CR3. Experimental hematomas resolved much more slowly after the injection of whole blood than after the injection of RBC. Qualitative microglial and astrocytic responses were quite similar. However, at 48 h, iron- and ferritin-reactive microglia were more numerous following the injection of whole blood. After injections of either type, ferritin-immunoreactive cells were more abundant than iron-positive cells. This observation implied that the biosynthesis of holoferritin protein and iron incorporation proceeded independently. Expression of CR3 on the surface of microglia was much more prominent after whole blood, suggesting a role of inactivated complement 3b in the attraction of additional phagocytes. Conversion to hemosiderin began at 5 days after the injection of either blood or RBC. The lesions caused initial destruction of astrocytes in the perifocal zone as judged by GFAP- and FRP-immunoreactivity. However, at 5 days, astrocytic processes reentered the perifocal zone and intermingled with microglia and macrophages. It is proposed that this contact between astrocytes and microglia reversed the uncoupling of ferritin biosynthesis and iron incorporation and initiated the storage of iron and formation of hemosiderin.

Triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) induces nuclear translocation of NF-kappa B (p50/p65) in human monocytes and enhances viral replication in HIV-infected monocytic cells.

Monocyte/macrophages may harbor HIV in a nonproductive fashion for prolonged periods of time. Viral gene expression may be reactivated by stimulation of the cells with LPS or cytokines such as TNF-alpha in vitro. The effect of LPS and TNF-alpha is mediated by their ability to induce nuclear translocation of the DNA-binding heterodimer NF-kappa B (p50/p65), which binds to a specific sequence in the HIV-long terminal repeat. The present study demonstrates that triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) enhances viral replication in HIV-infected human monocytic cells. Monocytic cell lines and normal peripheral blood monocytes were infected with HIV-1 in vitro and cultured in the presence or absence of F(ab')2 fragments of monoclonal anti-CR1 or anti-CR3 Abs or with C3 fragments. Stimulation of CR1 or CR3 induces a two- to fourfold increase in the amount of cell-associated and released p24 Ag in cell cultures that was equivalent to that observed in control cultures triggered with LPS. We further observed that stimulation of CR1 or CR3 induces the nuclear translocation of NF-kappa B p50/p65 in infected cells. Translocation of NF-kappa B p50/p65 was also observed following stimulation of CR1 or CR3 of uninfected peripheral blood monocytes from HIV-seronegative donors. The amount of protein translocated was similar to that observed when cells were stimulated with rhTNF-alpha. TNF-alpha did not mediate the translocation of NF-kappa B p50/p65 induced by triggering of complement receptors. Taken together, these observations suggest that HIV gene expression may be activated in infected monocytes through interaction of the cells with complement-opsonized particles and that enhanced viral replication is associated with C3 receptor-mediated nuclear translocation of the NF-kappa B complex.

Elastin and collagen degradation products in urine of patients with cystic fibrosis.

Elastin degradation has been reported to be increased in patients with cystic fibrosis (CF). In order to further explore evidence for elastin degradation in a group of 18 patients with CF with a wide range of disease severity, we used an isotope dilution method to measure urinary desmosine (DES) and isodesmosine (IDES), amino acids derived exclusively from cross-linked elastin, and hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), amino acids derived exclusively from cross-linked collagen. Urinary DES and IDES (mean +/- SD) were 23.9 +/- 30.7 and 18.5 +/- 22.4 micrograms/g creatinine, respectively, in the patients with CF versus 7.5 +/- 1.7 and 6.8 +/- 1.4 micrograms/g creatinine, respectively, in 10 healthy control subjects (p < 0.001); only two patients with CF had DES values within the control range. The values of urinary HP and LP in the CF group were 54.9 +/- 39.1 and 12.3 +/- 8.6 nmol/mmol creatinine, respectively, versus 24.5 +/- 5.8 and 5.1 +/- 2.7 nmol/mmol creatinine, respectively, in the controls (p < 0.005). Both HP and LP were highly correlated (r = 0.71, p < 0.0001). Patients with CF had active pulmonary inflammation; neutrophils were abundant in the bronchoalveolar lavage fluid of the CF group and correlated with elastase activity measured with methoxysuccinyl Ala-Ala-Pro-Val paranitroanilide (r = 0.61, p < 0.05). Airway neutrophils had decreased expression of the complement receptor CR1 (CR1/CR3 of 0.17 +/- 0.15 versus 1.0 for blood neutrophils), a change known to be caused by uninhibited neutrophil elastase. We conclude that lung elastin is the most likely source of the increased DES and IDES in CF.

Amoeboid microglial response following X-ray-induced apoptosis in the neonatal rat brain

The phagocytic response following X-ray-induced apoptosis in the neonatal rat brain was examined by immunohistochemistry with the antibodies OX-6 and OX-42 which recognize MHC class 1T antigens and the CR3 complement receptor, respectively. Few OX-6-immunoreactive cells were observed in control rats, and in rats irradiated at postnatal day 2 and examined during the first 2 postnatal weeks. However, a transient increase in the number of OX-42-immunoreactive amoeboid microglia, containing large numbers of apoptotic cells, occurred at 6, 24 and 48 h after irradiation when compared with age-matched controls. These results show that X-ray-induced apoptosis promotes a short-lasting phagocytic response.

Postmortem delay effects on neuroglial cells and brain macrophages from Lewis rats with acute experimental allergic encephalomyelitis: an immunohistochemical and cytochemical study

The effects of increasing postmortem delay (PMD) times on morphological, immunological and functional characteristics of various brain cells both in situ and in vitro were studied in postmortem brain tissue derived from rats with acute experimental allergic encephalomyelitis (EAE). A decline of the brain tissue structure was first noted after a PMD of 6 h. Radial glia in the cerebellum were frequently interrupted and retractions artifacts appeared around brain cells. However, even after the longest PMD interval of 18 h the quality of the cell and tissue structure was still good enough for immunohistochemical characterization. Immunohistochemical staining of frozen and fixed rat brain tissue sections resulted in an enhancement of the immunoreactivity after a PMD of 4 h, using a panel of mono and polyclonal antibodies directed against glial fibrillary acidic protein (GFAP), basement membranes (laminin), brain macrophage antigens (EDI and ED2), and various immunologically important surface molecules, such as major histocompatibility complex (MHC) class II (Ia) antigen (OX6), CR3 complement receptor (ED8), and leukocyte common antigen (OX1). No increase in staining intensities with the EDI, ED8 and OX6 mAbs specific for macrophage antigens could be detected on brain macrophages that were isolated from brain tissue of rats with EAE obtained after various PMD intervals. Irrespective of the PMD interval, viable astrocyte cell cultures were obtained with comparable staining intensities for GFAP. These cultured astrocytes were capable of ingesting Latex beads and were highly proliferative as measured by BrdU uptake, at all investigated PMDs. Thus, even after long PMD intervals, brain material can be used successfully. Other data suggest that the situation is similar to human brain material, even though the PMD times may be somewhat different.

Distinct Tyrosine Kinase Activation and Triton X-100 Insolubility upon FcγRII or FcγRIIIB Ligation in Human Polymorphonuclear Leukocytes.: IMPLICATIONS FOR IMMUNE COMPLEX ACTIVATION OF THE RESPIRATORY BURST

Two tyrosine kinase-dependent pathways exist for activation of the respiratory burst by polymorphonuclear leukocyte (PMN) immunoglobulin G Fc receptors. Direct ligation of Fc gamma RII activates the respiratory burst, but ligation of the glycan phosphoinositol-linked Fc gamma RIIIB does not. Instead, this receptor and the integrin complement receptor CR3 synergize in activation of the respiratory burst (Zhou, M.-J., and Brown, E. J. (1994) J. Cell Biol. 125, 1407-1416). Here we show that direct ligation of Fc gamma RII leads to activation and Triton X-100 insolubility of the Src family kinase Fgr, without effect on the related myeloid Src family member Hck. In contrast, adhesion of PMN via Fc gamma RIIIB leads to activation and Triton X-100 insolubility of Hck but not Fgr. The exclusive association of Fc gamma RIIIB with Hck activation and Triton insolubility is not solely a result of its glycan phosphoinositol anchor, since decay accelerating factor (CD55), another prominent glycan phosphoinositol-anchored PMN protein, is associated with Fgr insolubility to a greater extent than Hck. Ligation of decay accelerating factor, with or without coligation of CR3, does not activate the PMN respiratory burst. Coligation of Fc gamma RIIIB with Fc gamma RII overcomes the pertussis toxin inhibition of H2O2 production in response to direct ligation of Fc gamma RII. These data support the hypothesis that activation of Hck upon Fc gamma RIIIB ligation has a role in generation of the synergistic respiratory burst.

Comparison of the binding of C3S and C3F to complement receptors types 1, 2, and 3.

The two common polymorphic variants of C3 are C3S/HAV4.1- and C3F/HAV4.1+. It was reported previously that erythrocytes coated with C3F rosetted more strongly with mononuclear cells than erythrocytes coated with C3S. We examined the binding of C3S/HAV4.1- and C3F/HAV4.1+ to complement receptors CR1, CR2, and CR3. The binding of 125I-labeled C3b dimers to erythrocyte CR1 was measured. Scatchard analysis showed a two-binding constant model with very similar binding constants for dimers prepared with C3S and C3F: for C3S Kd1 = 18.3 +/- 2 nM; Kd2 = 6.2 +/- 1 nM; for C3F Kd1 = 21.5 +/- 4 nM; Kd2 = 7.2 +/- 3 nM (mean +/- SEM). One-third of the binding sites were of the higher affinity. The rosetting of erythrocytes with different densities of iC3b, prepared from C3S or C3F, to CR2 on Raji cells was analyzed. The percentage of Raji cells rosetted was related to the coating dose of EC3bi: 400 molecules/cell = 9% rosettes; 5000 molecules/cell = 75%. The dose-response curves were very similar for C3S- and C3F-coated erythrocytes. CR3-dependent rosetting was studied in a similar manner by using neutrophils activated with f-met-leu-phe. CR3-dependent rosette formation with the indicator erythrocytes (600 to 6000 iC3b/cell) increased from 10% to 60% in a dose-dependent manner and was closely similar for C3S and C3F. Inhibition of CR2 and CR3 rosetting by fluid phase ligand was also studied. iC3b dimers (0.4 to 50 micrograms/ml) inhibited CR2-dependent rosetting in a dose-dependent manner but had no inhibitory effect on CR3-dependent rosetting. When the dimers were absorbed to fluorescent microspheres, they mediated phagocytic uptake of the microspheres in a CR3-dependent manner by neutrophils. CR3-dependent binding by activated neutrophils required surface-bound ligand.

Targeting of an anti-CR3 (CD11b/CD18) monoclonal antibody to spleen but not brain, in vivo in mice.

Monoclonal antibodies against the complement receptor CR3 (CD11b/CD18) interfere with leukocyte recruitment and have potential as immunotherapeutics. However, the pharmacokinetic behavior as well as the tissue uptake and organ distribution of antibodies against CR3 are largely unknown. The present study examined the organ uptake (brain, heart, lung, liver, kidney, and spleen) and the pharmacokinetics of the anti-CR3 antibody, 5C6, and its isotype control, rat IgG2b, in mice. The clearance of 5C6 was biexponential with half-times of 6.4 +/- 2.4 min and 2.9 +/- 0.9 hours. The clearance of the IgG2b was also biexponential with half-times of 27.0 +/- 12.8 minutes and 36.1 +/- 1.2 hours. The uptake of 5C6 by the brain was negligible even 24 hours after injection, and there was only a minor uptake by heart and kidney. The uptake of 5C6 by spleen at 2 hours after injection was 44.7 +/- 26.6% of injected dose per gram tissue, and this value was 60-fold greater than the uptake by liver at 2 hours. The early and selective uptake by spleen indicates that 5C6 may be utilized for delivery of therapeutics to this organ.

Early 72-kDa heat shock protein induction in microglial cells following focal ischemia in the rat brain

Focal cerebral ischemia in the adult rat produces induction of 72-kDa heat shock protein (HSP-72) in neurons, glia and endothelial cells. Double antigen immunocytochemistry was carried out to find out whether microglial cells express HSP-72 following 1-h middle cerebral artery (MCA) occlusion. A monoclonal antibody against the CR3 complement receptor (OX-42) specific for microglia was used followed by a monoclonal antibody against HSP-72. Co-localization of these antibodies was seen in cells of the ipsilateral corpus callosum and striatum at 3 h following 1-h MCA occlusion, and in the ipsilateral striatal penumbra, corpus callosum and cortex at 8 h. Results demonstrate that stellate microglial cells show an early response to 1-h MCA occlusion by expressing inducible HSP-72, thus suggesting that microglial cells are sensitive to the ischemic insult.

C3b covalently associated to tetanus toxin modulates TT processing and presentation by U937 cells

Complement protein C3, like C4 and α 2-macroglobulin (α 2M), is a potentially bivalent ligand: (1) its proteolytic fragment, C3b, is able to interact covalently with antigens, and (2) this bound fragment is able to interact non-covalently with specific complement receptors of antigen presenting cells (APC). The formation of antigen-C3b complexes frequently occurs in vivo at inflammatory sites during the early stages of an immune response. Tetanus toxin (TT)-C3b covalent complexes, prepared from purified proteins, were used to study how C3b association influences the handling of TT by U937 cells used as APC. TT-specific T cell proliferation following TT-C3b processing was observed at a concentration when TT alone was inefficient. Whereas TT pinocytic uptake was low, TT-C3b uptake, through the help of complement receptor CR1, was three times higher. Free TT was rapidly transported to the lysosomes where it was proteolysed, whereas TT-C3b complexes were first retained in the endosomes and underwent only limited proteolysis. While the ester link of the complexes was fairly stable in the endosomes, it was gradually hydrolysed in the lysosomes with ensuing efficient proteolysis of the two proteins. This reflects the fact that associated C3b escorts TT during intracellular trafficking in the APC, and influences antigen processing. A triple role of C3b escorting antigen resides at the level of antigen uptake, routing, and proteolysis inside U937 cells, thus modulating antigen-dependent T cell proliferation.

The structure of an alternate form of complement C3 that displays costimulatory growth factor activity for B lymphocytes.

In this study, the structure of a novel 1.9-kb transcript coding for complement component 3 (C3) is described. This alternate C3 is identical to the 3' end of the C3 message beginning at position 3300 of the C3 cDNA. Its transcription appears to be driven by an alternate promoter located within intron 8 of the C3 gene. This alternate C3 message contains an open reading frame that may encode a 536-amino acid-long protein identical to the 3' part of the C3 alpha chain. The resulting protein contains the complement receptor CR2 binding site. The suggested 5' end of coding region of the alternate C3 includes information for a potential hydrophobic leader peptide that would allow secretion of the protein. In vitro assays with macrophage-depleted mouse splenic B cells indicate that an activity is secreted from cell lines transfected with the alternate C3 cDNA. Together with Sepharose-bound immunoglobulin M-specific monoclonal antibodies and interleukin 2, it costimulates the proliferation of B cells. Implications for possible in vivo functions are discussed.

Quantitative analysis of C4b dimer binding to distinct sites on the C3b/C4b receptor (CR1).

The complement receptor CR1 (CD35) is a transmembrane protein composed in its extracellular portion of short consensus repeats (SCR 1-30) organized into four long homologous repeats (LHR-A, LHR-B, LHR-C, and LHR-D). Each LHR, except LHR-D, contains a binding site for C3b and/or C4b within its first four SCR. The binding reaction between CR1 and soluble dimers of C4b (C4b2) was analyzed using the native receptor on human erythrocytes and full-length recombinant CR1 expressed in stably transfected Chinese hamster ovary (CHO) cells. CR1 mutants expressed similarly were used to determine the SCR of LHR-A required for C4b2 binding and the potential of C3b binding sites in CR1 to bind C4b2. Erythrocyte CR1, CHO cells expressing full-length recombinant CR1 (ABCD), constructs ACD, and SCR(1-4)D each bound C4b2 with similar affinities (Kd, approximately 4 x 10(-7) M). Construct SCR(1-2)D bound C4b2 with lower affinity (Kd, 1.4 x 10(-6) M) indicating that SCR(1-4) are required for a fully functional C4b2 binding site. Construct SCR(15-18)D, which contains a C3b site, also bound C4b2 with lower affinity (Kd 1.2 x 10(-6) M) than its binding to C3b dimers. Constructs SCR(15-16)D and D did not bind C4b2. Each CR1 construct that bound C4b2 functioned as a cofactor for factor I-mediated cleavage to C4d.

Viral antibodies in infectious mononucleosis

Patients with Epstein-Barr virus (EBV) infectious mononucleosis (IM) usually develop heterophilic antibodies and some autoantibodies. Antibodies to rubella, measles, adeno-, entero-, herpes simplex, cytomegalo- and varicella-zoster viruses were titrated in sera from IM patients and matched healthy controls using the complement fixation test (CFT) and the haemagglutination inhibition test. Except for herpes simplex virus and cytomegalovirus, the IM sera had significantly higher arithmetical and geometrical mean antibody titres and showed in most cases higher antibody prevalences in the CFT. The titre rise was most pronounced for rubella and measles antibodies, between 2- and 3-fold. There were no cases of very high titres occasionally seen in IM. The IM sera had higher total IgG serum levels than the controls, 17.27 g/l and 11.8 g/l, respectively (P < 0.001). The present data show that in addition to previously reported high levels of some autoantibodies and of heterophilic antibodies, there is a more general increase in IgG antibodies to commonly occurring viruses. This increase is most likely due to the polyclonal activation of B-lymphocytes following the binding of EBV to the complement receptor CR2 (CD21). When due consideration is given to the possible occasional occurrence of a false positive rubella IgM test, the raised antibody-titres will most likely not interfere with routine diagnostics.

Activation of polymorphonuclear neutrophilic granulocytes following burn injury: alteration of Fc-receptor and complement-receptor expression and of opsonophagocytosis.

Polymorphonuclear neutrophilic leukocytes (PMNLs) play a key role in host defense, and phagocyte dysfunction has been associated with increased susceptibility to infection in patients with thermal injury. We have used flow cytometric analysis (FCM) to longitudinally study PMNL expression of IgG Fc-receptor II (Fc gamma RII) and Fc-receptor III (Fc gamma RIII), as well as the complement receptors CR1 (receptor for C3b) and CR3 (receptor for C3bi) in 22 patients with large burns. Analyses of PMNL complement and immunoglobulin-mediated phagocytosis of Candida albicans were performed in parallel. Burn patient PMNL Fc gamma RIII expression was decreased to 58% of control values at admission, and remained low for the first 3 weeks. The expression of patient PMNL Fc gamma RII was not altered at admission or throughout the hospital stay. The CR1-dependent fluorescence was increased by 62% at admission, and reached a maximum at day 2, 138% greater than that of controls. The CR1 expression then gradually returned to normal at discharge. The PMNL CR3-dependent fluorescence showed an increase of 110% at admission and remained high during the first 3 weeks. The immunoglobulin-mediated phagocytosis was decreased by 12% at admission, whereas the lowest value was observed at day 10, with a reduction of 30% compared with controls. The patient PMNL complement-mediated phagocytosis of C. albicans was increased by about 160% at admission, and reached a maximum at day 2, before it gradually decreased to control levels at discharge. The expression of complement receptors correlated positively, whereas the expression of Fc gamma RIII correlated negatively, with total body surface area (TBSA) burn.(ABSTRACT TRUNCATED AT 250 WORDS)

Human monocyte infection by Leishmania (Viannia) panamensis. Role of complement receptors and correlation of susceptibility in vitro with clinical phenotype.

Peripheral blood monocytes (PBMs) from healthy individuals who had experienced distinctive clinical outcomes after natural infection with Leishmania (Viannia) were evaluated in vitro with respect to susceptibility to infection by stationary phase promastigotes of L. (V). panamensis. Concomitantly, the role of complement receptors (CR) CR1 and CR3 in the attachment and entry of L. (V). panamensis into human monocytes was analyzed using mAbs to CR1 (CD35) and CR3 (CD11b) to inhibit competitively these early events in the host-parasite interaction. Cell adherence to fibronectin was examined to determine how modulation of CR activity affected the attachment and uptake of this parasite species. The human monocyte cell line U-937 was also evaluated and found to provide a reproducible control for L. (V). panamensis infection in vitro. Opsonization with fresh AB+ serum markedly enhanced uptake by both PBMs and U-937 cells, and the fluid phase blocking of CR1 and CR3 resulted in partial inhibition of attachment and/or internalization. Uptake rather than attachment was abrogated by antireceptor antibodies in PBMs from previously infected individuals, whereas attachment was diminished in PBMs from unexposed controls. Adherence of PBMs to fibronectin resulted in decreased infection. PBMs from persons who had experienced chronic disease 5 to 8.4 yr before these studies were significantly more susceptible to in vitro infection by L. (V). panamensis than PBMs from asymptomatically infected or control individuals based on the percentage of cells infected, the number of parasites per cell, and viability of intracellular parasites at 48 h postinfection. Neither blocking of CR nor modulation by fibronectin altered the pattern of susceptibility of PBMs from the different clinical groups. These findings provide evidence for the participation of CR in the infection of human monocytes by L. (V). panamensis and demonstrate a correlation between clinical phenotype and in vitro infection of PBMs cultured in the presence of autologous plasma before experimental infection.