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PubMed1984

High resolution isoelectric focusing of immunoprecipitated proteins under denaturing conditions. A simple analytical method applied to the study of complement component polymorphisms.

A simple analytical method for the study of structural protein polymorphisms is described. It consists of the immunoprecipitation of non-radiolabeled proteins using monospecific polyclonal antibodies followed by isoelectric focusing (IEF) under completely denaturing conditions in vertical polyacrylamide slab gels. The method uses small amounts of sample (usually unfractionated plasma or serum), requires no sophisticated equipment and allows the screening of large numbers of samples with comparatively small effort. This method has been applied in the identification of 2 human complement-component polymorphisms, C4-binding protein (C4-bp) and factor H (beta 1H).

Antigen-Antibody Reactions↗
PubMed1993

Separation of different forms of the fourth component of human complement by fast protein liquid chromatography.

Disruption of the thiolester in native C4 yields a 'C4b-like C4' molecule (iC4) that functionally resembles C4b and is therefore probably accompanied by conformational changes in the C4 molecule. In most purified C4 preparations, iC4 and C4b are present to a variable extent. In this study we evaluated the use of fast protein liquid chromatography (FPLC) to resolve and isolate these various forms of C4. C4 was purified from fresh human plasma in a 4-step procedure that included barium citrate adsorption, polyethylene glycol 6000 (PEG) precipitation, Q-Sepharose Fast Flow and mono Q ion exchange chromatography. The final preparation appeared to be homogeneous on SDS-PAGE and under reducing conditions consisted of three bands that corresponded to the intact alpha, beta and gamma chains of C4. In some preparations the alpha' chain of C4b was also observed. On a Mono Q column the purified C4 preparations could be separated into three peaks that by hemolytic assay and SDS-PAGE were characterized as representing native C4, and monomeric and dimeric iC4 (or monomeric and dimeric C4b). Finally, the apparent KA of the various forms of C4 for C4b-binding protein (C4BP) was investigated. The monomeric iC4 and C4b species demonstrated similar C4BP binding affinity with an apparent KA of 5.6-6.4 x 10(8) M-1, whereas their dimeric forms demonstrated a higher affinity for C4BP with an apparent KA: 0.9-2.3 x 10(9) M-1. Binding of native C4 to C4BP was undetectable.

Animals↗
PubMed1988

Characterization of immune complexes detected by the 125I-C1q binding assay in breast cancer.

The aim of the present study was to isolate and characterize immune complexes measured by the 125I-C1q binding assay in breast cancer sera. The C1q binding assay detects immune complexes by their binding to 125I-C1q and subsequent precipitation with PEG. We purified the C1q binding material from these precipitates by superose 6 gel filtration and cation exchange chromatography. The isolation steps were monitored by the C1q binding assay and the purified material was analyzed by SDS--PAGE and immunodiffusion. The C1q binding material isolated from two breast cancer sera contained complexes of IgM, C4b binding protein (C4-bp), and C1q. The C4-bp fraction showed significant C1q binding activity which increased after the addition of IgM. This C4-bp was not different from C4-bp isolated from normal serum. The IgM fraction, however, differed from normal IgM by its binding to C4-bp and C1q and its stronger affinity to the cation exchange column. These unique properties were not due to rheumatoid factor activity. They might be caused by a different glycosylation pattern or by complex formation with an as yet unknown polysaccharide antigen.

Antibodies, Neoplasm↗
PubMed1990

Immunology of atherosclerosis: cellular composition and major histocompatibility complex class II antigen expression in aortic intima, fatty streaks, and atherosclerotic plaques in young and aged human specimens.

There is evidence that fatty streaks in arteries can transform into atherosclerotic plaques. Mononuclear cells, including both monocytes and lymphocytes, are among the first cells participating in the development of atherosclerosis of experimental animals. To investigate the roles of different cell types in human atherosclerosis, we enumerated and compared the cellular compositions of normal intima, the transition zone (the area between the normal intima and the core of fatty streaks), fatty streaks, and plaques in young (age 16-30 years) and aged (over 60 years) human specimens using double-staining immunofluorescence with a series of monoclonal and polyclonal antibodies. T lymphocytes, both T helper/inducer (70% of T cells) and T suppressor/cytotoxic (30%) phenotypes, were found in every stage of atherosclerosis, constituting 30 to 40% of total cells in fatty streaks and transition zones of young subjects, and occasionally even in normal intima. Seventy percent of these T cells were HLA-DR positive, which indicated that most of them were activated. Macrophages were most frequent in fatty streaks and around the necrotic core of plaques. Smooth muscle cells, increasing from 5 to 30% with lesion progression, were HLA-DR positive where activated T helper cells occurred in the vicinity. The intracellular presence of the invariant gamma chain confirmed that HLA-DR was actually synthesized by these smooth muscle cells. Endothelial cells were HLA-DR positive above those regions of the lesions where HLA-DR-positive cells had accumulated, but not in normal intima, again suggesting induction of HLA-DR expression by T-cell-derived gamma-interferon. Furthermore, most HLA-DR-positive cells were also identified as HLA-DP and HLA-DQ positive. This aberrant major histocompatibility complex class II antigen expression in smooth muscle and endothelial cells may participate in the perpetuation of the atherogenetic autoimmune reaction.

Adolescent↗
PubMed1992

Accelerated decay of the cell bound C4b2a complex by serum of patients with membranoproliferative glomerulonephritis and acute poststreptococcal glomerulonephritis.

Serum from patients with membranoproliferative glomerulonephritis (MPGN) and acute poststreptococcal glomerulonephritis (APSGN) accelerated the decay of the cell bound C4b2a (C42) and C4b hemolytic activity relative to pooled normal human serum (pNHS) after 5 min incubation at 30 degrees C in EDTA-GVB. The accelerated decay of the C42 hemolytic activity was heat stable (56 degrees C 30 min) and was inhibited by monoclonal antibody against human C4 binding protein (MoAb:C4BP) or C4 binding protein (C4BP) depleted serum. C4 nephritic factor (C4NeF) was employed to stabilize the labile classical pathway C3 convertase C42 complex. Serum from patients with MPGN and APSGN reduced the C4NeF stabilizing activity. Sera from 32 of 46 patients with MPGN and all of 7 patients with APSGN reduced the C42 hemolytic activity relative to 50 normal human serum (NHS) after 5 min incubation at 30 degrees C in EDTA-GVB, and there was no relationship with the serum concentration of C4BP. In vivo, accelerated decay of C42 convertase might interfere with the clearing and processing mechanism of circulating immune complexes (IC) by reducing deposition of C3b on the IC lattice.

Acute Disease↗
PubMed1993

Eosinophil major basic protein enhances the expression of neutrophil CR3 and p150,95.

BACKGROUND: We examined the effect of major basic protein (MBP) stimulation on the expression of the neutrophil B2-integrins: LFA-1 (CD11a/CD18), CR3 (CD11b/CD18), and p150,95 (CD11c/CD18). METHODS: Incubation of neutrophils with 0.75 to 3.0 mumol/L MBP for 30 minutes at 37 degrees C resulted in concentration-dependent increases in CR3 expression as detected by staining with the CD11b-specific monoclonal antibody, Leu-15. RESULTS: The expression of CR3 was significantly (p < 0.001) higher when neutrophils were stimulated with 1.5 mumol/L (53% +/- 9% increase) or 3.0 mumol/L (100% +/- 17% increase) MBP as compared with unstimulated neutrophils. The kinetics of MBP-stimulated CR3 expression were rapid and were similar to those of 100 nmol/L. N-formyl-methionyl-leucyl-phenylalanine-stimulated enhancement of CR3 expression. Incubation of neutrophils with reduced and alkylated MBP resulted in significantly lower (p < 0.05) increases in CR3 expression as compared with stimulation with native MBP. In addition, neither eosinophil cationic protein nor eosinophil-derived neurotoxin altered neutrophil CR3 expression. MBP stimulated minimal increases in LFA-1 expression. However, staining with the monoclonal antibody Leu-M5 (anti-CD11c) revealed that MBP stimulated significant increases in the expression of p150,95. CONCLUSIONS: These results indicate that MBP stimulates the increased expression of neutrophil adhesion molecules, which in turn may enhance the inflammatory role of neutrophils in the late-phase events of allergic diseases.

Blood Proteins↗
PubMed1989

Cell to cell contact enhances the proliferation of acute myeloid leukemia (AML) cells in vitro without an apparent role of adhesion glycoproteins LFA1, MAC1 and P150-95.

Acute myeloblastic leukemia (AML) cells were cultured under conditions facilitating or preventing cell to cell contact. Proliferation of AML blasts of 24 patients was assessed in semisolid (containing 0.9% methylcellulose) and in liquid cultures, in which identical concentrations of colony stimulating factors (CSFs) had been provided. In all but one of the cases, significant DNA synthesis (evaluated by 3H-thymidine uptake) was observed when the cells were incubated in the liquid system, whereas in only 14 cases (58%), the cells were able to form clusters or colonies in the semisolid system. These findings suggest that AML cells from a large proportion of patients can proliferate only after stimulation with CSFs in a liquid system, i.e. when cultured under conditions permitting reciprocal contact between the cells. To establish further the importance of cell to cell contact for AML cell proliferation, cells from 19 patients were cultured in liquid medium concurrently in flat bottom microwells (in which a majority of the cells lie separate) and in round bottom microwells (in which cells show a tendency to aggregate). A significantly higher 3H-thymidine (TdR) incorporation in the round bottom cultures was observed in 15 out of 19 cases. The role of the leukocyte function antigens (LFA) LFA1, Mac1, P150-95 in this phenomenon was then analyzed, as these structurally related glycoproteins are involved in reactions requiring contact between hematopoietic cells. Membrane expression of the three antigens was first examined in 16 patients. LFA1, Mac1 and P150-95 were expressed on the AML cells of 15, 6 and 8 patients, respectively. Expression of these antigens did not change following short term incubation of the AML cells in the presence of CSFs. AML cells cultured in presence of saturating concentrations of monoclonal antibodies reacting with structures of these 3 antigens in order to abrogate their function did not suppress 3H-TdR uptake. Thus, no direct role for LFA1 and/or Mac1 and/or P150-95 antigens in mediating contact-induced AML proliferation could be demonstrated. It remains to be established which components are involved in the cell-cell contact-mediated upregulation of AML cell proliferation.

Antigens, CD↗
PubMed1985

Structural and functional similarities between the major hemolymph protein of fall armyworm and cat C4 binding protein of the complement system.

We have investigated the structural and functional similarities between insect (armyworm) major larval serum protein (LSP-I) and mammalian (Cat) C4 binding protein (i) by comparing the major chymotryptic peptide patterns using sodium dodecyl sulfate polyacrylamide gel electrophoresis and (ii) their ability to prevent assembly of classical C3 convertase (EAC142). Both Cat C4 bp and Spodoptera LSP-I are composed of identical subunits of Mr = 75,000 and Mr = 80,000, respectively and generate the characteristic cleavage product of Mr = 50,000 on limited proteolysis. These observations are interpreted as an indication that the major hemolymph protein (LSP-I) of fall armyworm is a homologue of the C4bp of the mammalian complement system.

Amino Acids↗
PubMed1995

The leukocyte integrin gene CD11c is transcriptionally regulated during monocyte differentiation.

The leukocyte integrins, LFA-1, Mac-1 and p150,95, are heterodimeric proteins that consist of a distinct alpha and a common beta subunit. The beta subunit gene (CD18) is constitutively expressed on all leukocytes, however, the alpha subunit genes for LFA-1, Mac-1 and p150,95 (CD11a, CD11b and CD11c, respectively) show cell- and developmental stage-specific expression. We investigated the regulation of the CD11c gene in the promyeloblastic leukemic cell line, HL60, following differentiation along the monocytic pathway with phorbol 12-myristate 13-acetate (PMA). The steady-state level of CD11c mRNA increased markedly over 48 hr from the undetectable level present before differentiation. The half-life of CD11c MRNA in differentiated HL60 cells was not unusually long and similar to that of CD18 mRNA found in both undifferentiated and differentiated cells which suggested that altered mRNA stability did not account for the appearance of CD11c mRNA. Nuclear run-on analysis revealed that transcriptional activation during differentiation resulted in the appearance of CD11c mRNA. Inhibition of protein synthesis by cycloheximide in undifferentiated HL60 cells did not result in transcriptional activation of the CD11c gene. However, there was a significant increase (approximately eight-fold) in the steady-state level of CD18 mRNA which was not the result of transcriptional activation. Inhibition of protein synthesis in differentiated HL60 cells did not lead to significant changes in the steady-state levels of either CD11c or CD18 mRNAs. These findings indicated that the CD11c gene is regulated by transcriptional mechanisms which require prior protein synthesis. Transcriptional activation of the CD18 gene as a result of differentiation with PMA also requires protein synthesis. Further, in the absence of protein synthesis in undifferentiated HL60 cells, post-transcriptional mechanisms stabilize CD18 mRNA.

Blotting, Northern↗
PubMed1996

Regulation of the leukocyte integrin gene CD11c is mediated by AP1 and Ets transcription factors.

The leukocyte integrin gene, CD11c, encodes the chi subunit of the p150,95 (CD11c.CD18) receptor. Expression of the CD11c gene is predominately seen in monocytes, but has also been detected in some B- and T-cell neoplasms and in some large-cell lymphomas of uncertain origin. To elucidate the molecular mechanisms that govern the expression of CD11c, we have cloned and characterized the promoter region of this gene. The DNase I footprint and mobility shift analyses revealed five sites within the -86 to +40 region that interact with nuclear proteins. The -62 to -44 region contains two consensus sequences for AP1 (referred to as AP1-1 and AP1-2) and were shown to bind purified c-jun protein. Co-transfection of c-fos and c-jun expression constructs with a CD11c promoter-CAT fusion into HL60 cells led to a 6.7-fold increase in CD11c promoter activity. We show that c-fos and c-jun mediate their effects through both AP1-1 and AP1-2 which function in an additive manner. Regions -42 to -34 and -13 to -5 contain consensus sequences for Ets factors (referred to as Ets C and Ets A, respectively). Deletion of Ets resulted in a significant reduction in phorbol ester-induced expression of CD11c, whereas deletion of Ets A led to only a modest loss in CD11c expression. We show that Ets C cooperates with the AP1 sites to regulate CD11c expression.

Base Sequence↗
PubMed1990

A novel population of cells in the peripheral blood of a cow with a neurofibrosarcoma.

An unusual population of leukocytes was observed in the peripheral blood of a cow with a large tumor burden, using flow microfluorimetry. This new population accounted for 50% of the total cells in the peripheral blood of this animal. These cells expressed the p150,95 molecule (bovine CD11c equivalent), identified by the monoclonal antibody C5B6, a molecule found on myeloid cells and activated lymphocytes. The new population did not express the pan T molecules BoCD2 (the bovine T11 equivalent), BoCD5 (the bovine CD5 equivalent) or surface IgM. Isolated peripheral blood mononuclear cells maintained in bulk culture were able to kill autologous tumor cells and BHV-1 infected A549 in an NK-like assay. In vitro cytotoxicity by cells cultured from the peripheral blood of this animal was augmented 2- to 4-fold by the addition of IL-2.

Animals↗
PubMed1989

A monoclonal antibody to CD11c antigen inhibits the production of superoxide anion induced by concanavalin A in PMA-differentiated U-937 cells.

We have examined the effect of different monoclonal antibodies (MAb) to the CD11/CD18 family of surface antigens on the production of superoxide anion by phorbol myristate acetate (PMA)-differentiated U-937 cells upon stimulation with concanavalin A (Con A). The anti-CD11c MAb HC1/1 displayed a significant inhibition, whereas MAb to the other members of the CD11 family did not show a relevant inhibitory effect. These findings suggest that the CD11c antigen is involved in the triggering of the respiratory burst response by mononuclear phagocytic cells.

Antibodies, Monoclonal↗
PubMed1982

Calcium-dependent aggregation of human serum amyloid P component.

Normal human serum or isolated human amyloid P component was ultracentrifuged on density gradients containing either 10 mM EDTA or different concentrations of Ca2+ between 0.15 and 2.15 mM. In the presence of Ca2+ concentrations of 1 mM or more human P component sedimented more rapidly than it did in the presence of lower Ca2+ levels or of EDTA. This phenomenon was due to Ca2+-dependent aggregation of P component molecules and did not require the presence of any other serum constituents. It was completely inhibited by incorporating a physiological concentration (40 mg/ml) of serum albumin in the gradients, suggesting that free ionized Ca2+ is required to promote aggregation of the P component. P component from the mouse and the plaice (Pleuronectes platessa L.), a marine teleost, did not undergo the same Ca2+-dependent aggregation as human P component. These observations resolve a discrepancy existing in the literature concerning the sedimentation rate of human P component in density gradient ultracentrifugation and shed new light on its behaviour with respect to Ca2+ which may be relevant to the deposition of P component in amyloidosis.

Amyloid↗
PubMed1982

A study of a covalent-like interaction between soluble nascent C4b and C4-binding protein.

In the classical pathway of complement, the interaction between C4b and C4bp can be considered as a control of the C3 convertase formation. Purified C4-binding protein (C4bp) interacts with soluble nascent C4b to form covalent-like complexes; the interaction is also possible with nascent C4b-like C4, but not with C4, C4b or C4b-like C4. Formation of the complexes upon incubation of C4bp, C4 and C1s appears to involve a single link between a subunit of C4bp and the alpha' chain of C4b, as observed by SDS-polyacrylamide gel electrophoresis in reducing conditions (160 000 dalton band). In non-reducing conditions, a mixture of C4b-C4bp complexes is observed as a function of the C4b:C4bp molar ratio, with apparent molecular weights differing by a value of 210 000 and reflecting different C4b-C4bp associations. A maximum of five molecules of C4b are bound per molecule of C4bp, which appears to consist of 10 subunits of apparent molecular weight 72 000. The link between C4b and C4bp is partially destroyed by 1 M hydroxylamine at pH 9.0; its formation is strongly inhibited by 3.5 mM hydroxylamine or 60 mM methylamine at pH 9.0. These findings suggest an ester or amide bond between the activated carboxyl group of the thioester bridge in the alpha' or alpha chain of nascent C4b or C4b-like C4 and a hydroxyl or amino group of C4bp. Thus, C4bp might compete with other C4b acceptors such as membranes or IgG.

Carrier Proteins↗
PubMed2005

Characterization of alphaX I-domain binding to Thy-1.

The beta2 integrins are found exclusively in leukocytes and they are composed of a common beta chain, CD18, and one of four unique alpha chains, CD11a (alphaL subunit), CD11b (alphaM subunit), CD11c (alphaX subunit), or CD11d (alphaD subunit). alphaX-beta2 which binds several ligands including fibrinogen and iC3b is expressed in monocytes/macrophages and dendritic cells playing an important role in the host defense. Despite the unique characteristics on expression and regulation, alphaX-beta2 is less functionally characterized than other beta2 integrins. To understand the biological function of alphaX-beta2 more, we tested the possibility that alphaX-beta2 binds Thy-1, a membrane protein involved in cell adhesion and signaling regulation in neurons and T cells. Here we report that a ligand binding moiety of alphaX-beta2, the I-domain, bound Thy-1 in a specific and divalent cation-dependent manner. The dissociation constant (K(D)) of alphaX I-domain binding to Thy-1 was 1.16muM and the affinity of the binding was roughly 2-fold higher than that of alphaM I-domain. Amino acid substitutions on the betaD-alpha5 of alphaX I-domain (D249, KE243/244) showed low affinities for Thy-1 while other point mutations on alpha3-alpha4 and betaE-alpha6 loops of I-domain did not, suggesting that Thy-1 recognizes the portion of a betaD-alpha5 loop, possibly alpha5 helix. Taken together, these results indicate that alphaX-beta2 specifically interacts with Thy-1. Additionally, kinetic analysis reveals a moderate affinity interaction in the presence of divalent cations. Given the reported role of Thy-1 in the regulation of T cell homeostasis and proliferation, it is tempting to speculate that alphaX-beta2 may be involved in Thy-1 function.

Animals↗