Specificity of human lymphocyte complement receptors.

Erythrocytes, bone marrow-derived lymphocytes, monocytes, and granulocytes were shown to have a receptor activity for C4. Theis C4 receptor activity was studied in relation to the previously identified C3b and C3d receptors. By assay for inhibition of rosette formation by fluid-phase complement (C), only two different lymphocyte C receptors were demonstrated. The immune adherence receptor, the only one of the two shared in common with erythrocytes, was specific for C4 or the C3c region of C3b, but was unreactive with C3d. The other lymphocyte receptor, the C3d receptor, was specific for C3d fragments, but would also react to a lesser extent with the C3d region of uncleaved C3b. ThC3d receptor did not react with either C3c or C4. This specificity of the C3d receptor allowed certain cells which contained only C3d receptors to form rosettes with EAC1-3b and EAC1-3d, but not with EAC14. However, because C3d receptors bound EAC1-3d or C3d fragments more firmly than they did EAC1-3b or C3b fragments, many other types of cells containing only C3d receptors, formed rosettes with EAC1-3d but not with EAC1-3b. Erythrocytes and those lymphocytes which contained only immune adherence receptors, formed rosettes with EAC14 and EAC1-3D but not with EAC1-3d. A double-label assay was devised for the simultaneous detection of both types of C receptors on individual lymphocytes. This assay involved fluorescence labeling of one of the two C receptors with soluble C fragments in combination with the usual rosette method for labeling the other type of C receptor. With this double-label assay, it was observed that the two different lymphocyte C receptors capped independently and thus were located on different molecules which could each move through the fluid membrane matrix independently of the other.

Identification of a C3bi-specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes.

Cells expressing a membrane C receptor (CR(3)) specific for C3b-inactivator- cleaved C3b (C3bi) were identified by rosette assay with C3bi-coated sheep erythrocytes (EC3bi) or C3bi-coated fluorescent microspheres (C3bi-ms). C3bi- ms, probably because of their smaller size, bound to a higher proportion of cells than did EC3bi. C3bi-ms bound to greater than 90 percent of mature neutrophils, 85 percent of monocytes, 92 percent of erythrocytes, and 12 percent of peripheral blood lymphocytes. Binding of C3bi-ms to neutrophils, monocytes, and erythrocytes was inhibited by fluid-phase C3bi, Fab anti-C3c, or Fab anti-C3d but was not inhibited by F(ab')(2) anti-CR(1) (C3b receptor) or F(ab')(2) anti-CR(2) (C3d receptor) nor by fluid-phase C3b, C3c, or C3d. This indicated that monocytes, neutrophils, and erythrocytes expressed C3bi receptors (CR(3)) that were separate and distinct from CR(1) and CR(2) and specific for a site in the C3 molecule that was only exposed subsequently to cleavage of C3b by C3b inactivator and that was either destroyed, covered, or liberated by cleavage of C3bi into C3c and C3d fragments. Lymphocytes differed from these other cell types in that they expressed CR2 in addition to CRa. Lymphocyte C3bi-ms rosettes were inhibited from 50 to 84 percent by F(ab')(2)-anti-CR(2) or fluid-phase C3d, whereas C3d-ms rosettes were inhibited completely by F(ab')(2) anti-CR(2), fluid-phase C3bi, or fluid- phase C3d. Thus, with lymphocytes, C3bi was bound to CR(3), and in addition was bound to CR(2) by way of the intact d region of the C3bi molecule. In studies of the acquisition of C receptors occurring during myeloid cell maturation, the ability to rosette with C3bi-coated particles was detected readily with immature low-density cells, whereas this ability was nearly undetectable with high density mature polymorphonuclear cells. This absence of C3bi binding to polymorphs was not due to a loss of the CR(3) but instead was due to the maturation-linked acquisition of the abiity to secrete elastase that cleaved reagent particle-bound C3bi into CR(3)-unreactive C3d. Neither neutrophils nor monocytes bound C3d-coated particles at any stage of maturation. Assay of CR(3) with mature neutrophils required inhibition of neutrophil elastase with either soybean trypsin inhibitor or anti-elastase antibodies, and the amounts of these elastase inhibitors required to allow EC3bi rosette formation increased with neutrophil maturation. Because lymphocytes bound C3bi to CR(2) as well as to CR(3), specific assay of lymphocyte CR(3) required saturation of membrane CR(2) with Fab' anti-CR(2) before assay for rosettes with C3bi-ms. Only 3.5 percent of anti-CR(2)- treated peripheral blood lymphocytes bound C3bi-ms. Therefore, among normal blood lymphocytes the majority of the 12 percent C3bi-ms-binding cells expressed only CR(2) (8.5 percent), and the small proportion of C3bi-ms- binding cells that expressed CR(3) (3.5 percent) represented a distinct subset from the CR2(+) cells. Double-label assay indicated that 3.0 percent out of 3.5 percent of these CR(3)-bearing lymphocytes were B cells because they expressed membrane immunoglobulins. Of the remaining CR(3)(+) cells, 0.2 percent expressed either Leu-1 or 3A1 T cell antigens, and 0.6 percent expressed the OKM-1 monocyte-null lymphocyte determinant.

Characterization of the lymphocyte membrane receptor for factor H (beta 1H-globulin) with an antibody to anti-factor H idiotype.

Antibody to the binding site (idiotype) of anti-factor H was shown to have specificity for both B lymphocyte membrane H receptors and C3b. Goat F(ab')(2) anti-human H was purified by absorption and elution from H agarose and used for rabbit immunization to produce anti-anti-H (aaH). After absorption with nonimmune goat IgG, (125)I-labeled aaH bound to B lymphocytes and to sheep erythrocytes coated with C3b (EC3b) but did not bind to T lymphocytes or to EC3d. All B cell- and C3b-specific activities of the aaH were removed and subsequently recovered by absorption and elution of the antibody from either C3-agarose or goat-anti-H-agarose. This indicated that the aaH probably recognized a single common antigenic structure that was shared by anti-H, C3b, and the membranes of B cells. Affinity-purified aaH resembled H in that it bound to B cells, blocked the uptake of H onto B cell H receptors, and triggered B cells to release endogenous factor I (C3b inactivator). In addition, aaH functioned with factor I as either a cofactor for cleavage of fluid-phase C3b or a potentiator for cleavage of bound C3b. This same spectrum of C3 binding functions could not be demonstrated with either sheep anti-C3b or rabbit-anti-C3c. Analysis by sodium dodecyl sulfate- polyacrylamide get electrophoresis of the [(3)H]leucine intrinsically labeled B cell proteins reactive with the purified aaH revealed proteins of 100,000 M(r) and 50,000 M(r) without reduction, and after complete reduction of disulfide bonds, a single protein band of 50,000 M(r). This same protein molecular weight profile was also demonstrated with labeled B cell proteins that were absorbed and eluted from H-agarose. Thus, aaH is apparently specific for both B cell H receptors and C3b. However, because parallel analysis of C3b confirmed its known 115,000- and 75,000-M(r) polypeptide chain structure, the H receptor is probably not C3b and shares only the structure of the H binding site with C3b.

Release of endogenous C3b inactivator from lymphocytes in response to triggering membrane receptors for beta 1H globulin.

Human bone marrow-derived lymphocytes and cells from B lymphoblastoid lines were shown to have specific membrane receptors for beta 1H globin. Lymphocytes responded to the presence of beta 1H by releasing endogenously-synthesized C3b-inactivator. Very little spontaneous release of C3b-inactivator occurred in the absence of beta 1H. beta 1H-treated lymphocytes that either lacked complement receptor type one (CR1, the C4b-C3b receptor) or had their CR1 blocked with Fab'-anti-CR1 formed rosettes with C3b-coated sheep erythrocytes (EC3b) by adherence to complement receptor type two (CR2, the C3d-C3bi receptor). The mechanism of the beta 1H-induced EC3b rosette formation was shown to involve the release of lymphocyte C3b-inactivator that cleaved bound C3b into C3bi forming EC3bi. This lymphocytes-generated EC3bi then bound to CR2, forming rosettes. beta 1H-induced EC3b rosettes were completely inhibited by the presence of either anti-C3b-inactivator, F(ab')2-anti-CR2, Fab-anti-C3c, or Fab-anti-C3d, but were unaffected by the presence of fluid-phase concentrations of beta 1H up to 5.5 mg/ml or Fab'-anti-CR1. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography demonstrated that cell-free supernates of beta 1H-treated lymphocytes cleaved 3H-labeled C3b on EC3b into C3bi. Inhibition studies with chelating agents and sodium azide suggested that the release of C3b inactivator might involve a calcium and energy-dependent transport of this enzyme across the membranes of beta 1H-triggered cells. Because plasma beta 1H and C3b-inactivator are known to have important functions in the distinction of alternative pathway-activating substances from normal tissue, it is possible that this beta 1H receptor-C3b-inactivatory releasing system in lymphocytes may have an analogous function.

The sequential appearance of Ia-like antigens and two different complement receptors during the maturation of human neutrophils.

Ia antigens and two different types of complement (C) receptors appeared on membrane surfaces in a distinct sequence during the maturation of human neutrophils. Taking advantage of the finding that neutrophil celll density increased with maturation, density gradient centrifugation was used to separate neutrophils into fractions that were greatly enriched in cells representing individual stages of differentiation. Myeloblasts, the earliest cells recognized in the myeloid series of both normal and myelogenous leukemic individuals, expressed Ia determinants, whereas Ia determinants were absent or diminished on the majority of promyelocytes and completely undetectable on more mature granulocytes. Double marker studies demonstrated that Ia determinants were lost from the membrane of developing myeloid cells before the appearance of any type of C receptor. In the next phase of maturation defined by surface markers, neutrophils acquired a CR2-type C receptor (C3d receptor) that was similar in specificity to CR2 of B lymphocytes. This stage of maturation approximately corresponded to the myelocyte-metamyelocyte stage defined by standard morphologic criteria, and preceded the third stage of surface marker maturation when developing neutrophils began to express CR1-type C receptors (immune adherence, C4b-C3b receptors) in addition to CR2. In the final stage of surface marker-defined maturation, CR2 was lost from high density polymorphonuclear neutrophils and CR1 was maximally expressed. Normal blood polymorphonuclear neutrophils contained only 17% of CR2-bearing cells and these were shown to be of lower density than the majority of neutrophils that expressed only CR1. There was some variation in the correlation of surface marker expression and maturation stage defined by morphologic criteria, but in all cases the sequence of marker appearance was the same: Ia leads to CR2 leads to CR1CR2 leads to CR1.

Membrane receptors of mouse leukocytes. II. Sequential expression of membrane receptors and phagocytic capacity during leukocyte differentiation.

Analysis of four mature cell markers on mouse bone marrow leukocytes grown in vitro, demonstrated a distinct sequence of marker appearance during the terminal phases of granulocytic cell differentiation. A similar pattern of marker expression was also suggested by analysis of mature neutrophils and macrophages isolated from normal tissues. Among cultured neutrophils, receptors for the Fc portion of IgG (FcR) were first expressed on myelocytes and metamyelocytes, and then subsequently on more mature cells. Morphologically mature colony neutrophils (polymorphs) from agar cultures contained only FcR and complement receptor type two (CR(2)) (C3d receptor), and lacked both complement receptor type one (CR(1)) (C3b receptor) and the capacity to ingest latex, bacteria, or iron particles. Neutrophils from 2 and 3 wk liquid media cultures of marrow cells differed from agar grown neutrophils in that they had phagocytic capacity (particle ingestion) [Pi] in addition to FcR and CR(2). Furthermore, in the 4th and 5th wk of these continuous liquid cultures, CR(1) was also expressed, completing the surface marker profile of normal blood neutrophils. Based on these studies, the following order of appearance of these four markers on cells from the myelocytic series was proposed: FcR {arrow} FcR CR(2) {arrow} FcR CR(2) Pi {arrow} FcR CR(2) Pi CR(1). Differential studies of tissue leukocytes containing these same markers revealed that a heterogeneity existed among morphologically mature neutrophils. Even though 95 percent of blood polymorphs contained all four markers, the same was true of only half of spleen polymorphs and only 20 percent of bone marrow polymorphs. Cells of the monocyte-macrophage series were studies in parallel with neutrophils. Cultured marrow monocytes acquired the four mature cell markers so rapidly that the order of receptor appearance could not be determined. However, it was found that CR2 was lost during the terminal phase of monocyte maturation into activated macrophages.

Human megakaryocytes. I. Characterization of the membrane and cytoplasmic components of isolated marrow megakaryocytes.

Human marrow megakaryocytes have been isolated with high purity and yield by processing marrow cells sequentially through density centrifugation and velocity sedimentation. Analysis of the isolated cells for various platelet-associated components by immunofluorescence demonstrated that fibrinogen, plasma factor VIII antigen (factor VIII:AGN) platelet myosin, platelet glycoproteins I and III are present on the membrane and in the cytoplasm of over 90% of marrow megakaryocytes. Parallel studies of human and mouse megakaryocytes and platelets for IgG receptor (FcR), complement receptor type one (CR1) (C3b receptor), complement receptor type two (CR2) (C3d receptor), and Ia antigen by fluorescence and (or) rosette formation methods were performed. FcR were present on most human megakaryocytes and platelets. The Ia antigen was detected on a proportion (10-15%) of human megakaryocytes but it was undetectable on human platelets. CR1 was found on 20-40% of mouse megakaryocytes and also on a proportion of mouse platelets. These differentiation markers may be of use in monitoring megakaryocyte maturation.

Two different complement receptors on human lymphocytes. One specific for C3b and one specific for C3b inactivator-cleaved C3b.

IN THE PRESENT STUDY IT WAS SHOWN THAT NORMAL PERIPHERAL LYMPHOCYTES HAVE TWO DIFFERENT COMPLEMENT RECEPTORS: one for C3b (the immune adherence receptor) and one for C3b subsequent to its cleavage by C3b inactivator. The two receptors are not cross-reactive and were shown by tests with various antisera to be antigenically distinct. Both the immune adherence receptor and the receptor for C3b inactivator-cleaved C3b were found on normal peripheral lymphocytes and on cultured lymphoblastoid cells. In 15 out of 18 chronic lymphatic leukemia patients, the immune adherence receptor was either partially or completely missing from the peripheral lymphocytes, while the lymphocyte receptor for C3b inactivator-cleaved C3b was retained. Normal erythrocytes, on the other hand, were found to have only the immune adherence receptor. Granulocytes from normal peripheral blood appeared to have only a receptor for C3b and did not have a receptor for C3b inactivator-cleaved C3b.

Generation of three different fragments of bound C3 with purified factor I or serum. II. Location of binding sites in the C3 fragments for factors B and H, complement receptors, and bovine conglutinin.

The many different recognized functions of C3 are dependent upon the ability of the activated C3 molecule both to bind covalently to protein and carbohydrate surfaces and to provide binding sites for as many as eleven different proteins. The location of the binding sites for six of these different proteins (factors B and H, complement receptors CR(1), CR(2) and CR(3) and conglutinin) was examined in the naturally occurring C3-fragments generated by C3 activation (C3b) and degradation by Factor I (iC3b, C3c, C3d,g) and trypsin (C3d). Evidence was obtained for at least four distinct binding sites in C3 for these six different C3 ligands. One binding site for B was detectable only in C3b, whereas a second binding site for H and CR(1) was detectable in both C3b and iC3b. The affinity of the binding site for H and CR(1) was charge dependent and considerably reduced in iC3b as compared to C3b. H binding to iC3b-coated sheep erythrocytes (EC3bi) was measurable only in low ionic strength buffer (4 mS). The finding that C3c-coated microspheres bound to CR(1), indicated that this second binding site was still intact in the C3c fragment. However, H binding to C3c was not examined. A third binding site in C3 for CR(2) was exposed in the d region by factor I cleavage of C3b into iC3b, and the activity of this site was unaffected by the further I cleavage of iC3b into C3d,g. Removal of the 8,000-dalton C3g fragment from C3d,g with trypsin forming C3d, resulted in reduced CR2 activity. However, because saturating amounts of monoclonal anti-C3g did not block the CR(2)-binding activity of EC3d,g, it appears unlikely that the g region of C3d,g or iC3b forms a part of the CR(2)-binding site. In addition, detergent-solubilized EC3d (C3d-OR) inhibited the CR(2)-binding activity of EC3d,g. Monocytes and neutrophils, that had been previously thought to lack CR(2) because of their inability to form EC3d rosettes, did bind EC3d,g containing greater than 5 x 10(4) C3d,g molecules per E. The finding that monocyte and neutrophil rosettes with EC3d,g were inhibited by C3d-OR, suggested that these phagocytic cells might indeed express very low numbers of CR(2), and that these CR(2) were detectable with EC3d,g and not with EC3d because C3d,g had a higher affinity for CR2 than did C3d. A fourth C3 binding site for CR(3) and conglutinin (K) was restricted to the iC3b fragment. Because of simultaneous attachment of iC3b to phagocyte CR3 and CR(3), the characteristics of iC3b binding to CR3 could only be examined with phagocytes on which the CR(1) had been blocked with anti-CR(1). Inhibition studies with EDTA and N-acetyl-D-glucosamine demonstrated a requirement for both calcium cations and carbohydrate in the binding of EC3bi to CR3 and to K. However, CR(3) differed from K in that magnesium cations were required in addition to calcium for maximum CR(3) binding activity, and NADG produced less inhibition of CR(3) activity than of K activity.

Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells.

When phagocyte CR3 binds to iC3b on bacteria or yeast, phagocytosis and degranulation are triggered because of simultaneous recognition of iC3b via a CD11b I-domain binding site and specific microbial polysaccharides via a lectin site located COOH-terminal to the I-domain. By contrast, when phagocyte or natural killer (NK) cell CR3 adheres to iC3b on erythrocytes or tumor cells that lack CR3-binding membrane polysaccharides, neither lysis nor cytotoxicity are stimulated. This investigation showed that soluble CR3-specific polysaccharides such as beta-glucan induced a primed state of CR3 that could trigger killing of iC3b-target cells that were otherwise resistant to cytotoxicity. Anti-CR3 added before sugars prevented priming, whereas anti-CR3 added after sugars blocked primed CR3 attachment to iC3b-targets. Polysaccharide priming required tyrosine kinase(s) and a magnesium-dependent conformational change of the I-domain that exposed the CBRM1/5 activation epitope. Unlike LPS or cytokines, polysaccharides did not up-regulate neutrophil CR3 expression nor expose the mAb 24 reporter epitope representing the high affinity ICAM-1-binding state. The current data apparently explain the mechanism of tumoricidal beta-glucans used for immunotherapy. These polysaccharides function through binding to phagocyte or NK cell CR3, priming the receptor for cytotoxicity of neoplastic tissues that are frequently targeted with iC3b and sparing normal tissues that lack iC3b.

Interaction of platelet membrane receptors with von Willebrand factor, ristocetin, and the Fc region of immunoglobulin G.

The agglutination of human platelets by ristocetin and von Willebrand factor was inhibited by aggregated immunoglobulin (Ig)G and by Fc fragments of IgG, but not by Fab, F(ab')(2) or pFc fragments of IgG. Because this inhibition occurred with formalin-fixed platelets as well as with normal platelets, a generalized aggregation of fluid membrane components by Fc fragments was not responsible for this inhibition of ristocetin and von Willebrand factor-induced agglutination. Reciprocal inhibition of platelet Fc receptors was produced by prior incubation of platelets with von Willebrand factor and ristocetin. Sucrose density gradient ultracentrifugation studies demonstrated that aggregated IgG did not form fluid-phase complexes with von Willebrand factor and ristocetin. Furthermore, passage of von Willebrand factor and ristocetin through a column of immobilized heat-aggregated IgG did not alter platelet agglutinating activity which indicates that aggregated IgG did not inactivate von Willebrand factor or ristocetin. Thus, it was likely that the IgG-mediated interference with platelet agglutination by ristocetin and von Willebrand factor did not occur in the fluid phase but at the platelet surface. These studies suggest that the platelet membrane Fc receptor may be either a part of, or sterically related to, the membrane glycoprotein I complex that interacts with von Willebrand factor, and that occupation of one of these surface components blocks the availability of the other.

Surface markers of complement receptor lymphocytes.

Normal blood lymphocytes bearing complement receptors (CRL) were divided into two populations, one expressing both CR1 (C4b-C3b receptor) and CR2 (C3d receptor) and a second expressing only CR1. Nearly all of the population that expressed both CR1 and CR2 also bore membrane surface immunoglobulins (Ig) and Ia antigens. The majority of cells that had only CR1 lacked detectable surface Ig. These Ig- CR1+ CR2- cells could be distinguished from the majority of monocytes and immature granulocytes, in that the latter ingested latex particles and expressed CR2 as well as CR1. The Ig- CR1+ cells were further subdivided into an Ia-bearing subpopulation and another that lacked Ia. Among the Ig- Ia- CR1+ cells, one third formed spontaneous rosettes with sheep erythrocytes while all of the remaining CRL were erythrocyte-rosette negative. Essentially all CRL in normal blood had IgG Fc receptors, but a qualitative heterogeneity in the Fc receptors of Ia+ CRL vs. Ia- CRL was observed in their binding of different immune complex systems.

Combined studies of complement receptor and surface immunoglobulin-bearing cells and sheep erythrocyte rosette-forming cells in normal and leukemic human lymphocytes.

Human lymphocytes from normal peripheral blood, thymus, spleen, thoracic duct, and peripheral lymphocytes from patients with chronic lymphatic leukemia were studied for complement receptor sites (CRL), surface immunoglobulin (SIg), and for the ability to form rosettes with sheep erythrocytes (TRFC). The two B cell markers (CRL and SIg) were found to be in overlapping, but not totally identical populations, whereas cells that were able to form rosettes were found in a totally unrelated population of lymphocytes; TRFC is therefore probably a reliable marker for T cells. In peripheral blood 24% of lymphocytes had SIg, but only half of these were also CRL. Almost all of the non-SIg peripheral blood lymphocytes were TRFC. In the spleen and thoracic duct only a few lymphocytes were observed that had SIg and were not CRL. On the other hand, in two of three spleens studied 10-20% of cells were CRL that did not have SIg. In the thoracic duct all non-CRL that did not have SIg. In the thoracic duct all non-CRL, non-SIg cells were TRFC. In chronic lymphatic leukemia three findings were made: (a) The presence or absence of CRL was independent of the presence or absence of SIg so that in individuals whose cells were non-SIg. CRL were usually plentiful. (b) Leukemic cells were essentially negative for TRFC. (c) Leukemic cells reacted poorly with human C3 compared to mouse C3, EACmo detecting up to 20-fold more CRL than EAChu. This latter finding was in sharp contrast to normal CRL that reacted somewhat preferentially with EAChu. These data suggest that altered surface Ig receptors and complement receptors are present in chronic lymphatic leukemic cells. Since the cells obtained from all leukemic patients tested in this study had either the complement receptor or surface immunoglobulin in a high percentage of their cells and were essentially negative for TRFC, it is strongly suggested that leukemic lymphocytes are of B cell origin. The finding of lymphocytes with only one of the two B cell markers suggests that these markers are not uniformly present on all B cells and that depending on the source, one or the other may be deficient.

Neutrophil and monocyte cell surface p150,95 has iC3b-receptor (CR4) activity resembling CR3.

Previous investigations of p150,95 (CD11c), the third member of the CD18 membrane glycoprotein family that includes CR3 (Mac-1 or CD11b) and LFA-1 (CD11a), had demonstrated that solubilized p150,95 bound to iC3b-agarose in a manner similar to isolated CR3. The current study showed that membrane surface p150,95 also expressed iC3b-receptor activity and was probably the same as the neutrophil receptor for iC3b- or C3dg-coated erythrocytes (EC3bi or EC3dg) that had been previously designated CR4. Normal neutrophil and macrophage CR4-dependent EC3bi rosettes were inhibited by monoclonal anti-p150,95, and cells from a patient with CD18 deficiency did not form CR4-dependent EC3bi rosettes. With neutrophils that bore large amounts of CR1 and CR3 and little p150,95, EC3bi were found primarily via CR1 and CR3, and demonstration of p150,95-dependent rosettes required large amounts of fixed iC3b, low-ionic strength buffer, and antibody blockade of CR1 and CR3. By contrast, culture-derived macrophages expressed eight times more p150,95 than did monocytes and EC3bi were bound to both p150,95 and CR3 when EC3bi bore small amounts of fixed iC3b and assays were carried out in isotonic buffer. Comparison of the amounts of CR1, CR3, and CR4 in various tissues by immunoperoxidase staining revealed that CR4 was the most abundant C3 receptor molecule on tissue macrophages, and suggested that CR4 might be involved in clearance of C3-opsonized particles or immune complexes.

Regulation of the adhesion versus cytotoxic functions of the Mac-1/CR3/alphaMbeta2-integrin glycoprotein.

Mac-1/CR3 functions as both an adhesion molecule mediating the diapedesis of leukocytes across the endothelium and a receptor for the iC3b fragment of complement responsible for phagocytic/degranulation responses to microorganisms. Mac-1/CR3 has many functional characteristics shared with other integrins, including bidirectional signaling via conformational changes that originate in either the cytoplasmic domain or extracellular region. Another key to its functions is its ability to form membrane complexes with glycosylphosphatidylinositol (GPI)-anchored receptors such as Fc gammaRIIIB (CD16b) or uPAR (CD87), providing a transmembrane signaling mechanism for these outer membrane bound receptors that allows them to mediate cytoskeleton-dependent adhesion or phagocytosis and degranulation. Many functions appear to depend upon a membrane-proximal lectin site responsible for recognition of either microbial surface polysaccharides or GPI-linked signaling partners. Because of the importance of Mac-1/CR3 in promoting neutrophil inflammatory responses, therapeutic strategies to antagonize its functions have shown promise in treating both autoimmune diseases and ischemia/reperfusion injury. Conversely, soluble beta-glucan polysaccharides that bind to its lectin site prime the Mac-1/CR3 of circulating phagocytes and natural killer (NK) cells, permitting cytotoxic degranulation in response to iC3b-opsonized tumor cells that otherwise escape from this mechanism of cell-mediated cytotoxicity.

Transplantation studies in C3-deficient animals reveal a novel role of the third complement component (C3) in engraftment of bone marrow cells.

Mice deficient in complement C3 (C3(-/-)) are hematologically normal under steady-state conditions, and yet displayed a significant delay in hematopoietic recovery from either irradiation or transplantation of wild-type (WT) hematopoietic stem/progenitor cells (HSPC). Transplantation of histocompatible WT Sca-1(+) cells into C3(-/-) mice resulted in a (i) decrease in day 12 CFU-S, (ii) 5-7-day delay in platelet and leukocyte recovery, and (iii) reduced number of BM CFU-GM progenitors at day 16 after transplantation. Nevertheless, HSPC from C3(-/-) mice engrafted normally into irradiated WT mice, suggesting that there was a defect in the hematopoietic environment of C3(-/-) mice. Since C3(-/-) mice cannot activate/cleave C3, the C3 fragments C3a, C3a(des-Arg), and iC3b were examined for a role in HSPC engraftment. Liquid-phase C3a and C3a(des-Arg) increased CXCR4 incorporation into membrane lipid rafts (thus potentiating HSPC responses to SDF-1 gradients), whereas iC3b was deposited onto irradiated BM cells and functioned to tether CR3(CD11b/CD18)(+)HSPC to damaged stroma. The activity of C3a(des-Arg) suggested that C3aR(+)HSPC also expressed the C5L2 (receptor for C3a and C3a(des-Arg)) and this was confirmed. In conclusion, a novel mechanism for HSC engraftment was identified, which involves complement activation and specific C3 fragments that promote conditioning for transplantation and enhance HSPC engraftment.

Macrophage cytoskeleton association with CR3 and CR4 regulates receptor mobility and phagocytosis of iC3b-opsonized erythrocytes.

Alveolar macrophages (AM phi) were examined for CR1 (C3b receptor, CD35), CR3 (iC3b receptor; CD11b/CD18), and CR4 (iC3b receptor; CD11c/CD18) by assays for binding of C3-opsonized sheep erythrocytes (EC3b or EC3bi) and uptake of specific monoclonal antibodies (mAbs). In AM phi isolates from nine normal volunteers, 49% of cells bound EC3b and 71% bound EC3bi. Quantitation of receptors per cell with [125I]mAbs showed 8.5 x 10(4) CR4, 5.1 x 10(4) CR3, and 2.6 x 10(4) CR1. With most AM phi preparations, CR3 was the major receptor mediating attachment of EC3bi, despite the predominance of CR4 antigens. Anti-CR3 inhibited EC3bi rosettes by > or = 50%, whereas anti-CR4 blocked rosettes by < or = 18%. U937 cells differentiated with phorbol myristate acetate resembled AM phi in receptor expression but exhibited almost no CR4-dependent rosetting. Despite the relative inability of CR4 to mediate EC3bi attachment, AM phi ingestion of [51Cr]EC3bi was blocked by either anti-CR3 or anti-CR4. Two lines of evidence indicated that CR3 were more mobile within the membrane than were CR4. Immunofluorescence staining demonstrated patching and occasional capping of CR3, whereas CR4 remained uniformly distributed. This patching and capping of CR3 required the actin cytoskeleton, as it was inhibited by cytochalasin D. Modulation experiments using surfaces coated with anti-CR3 or anti-CR4 also showed that CR3 was more mobile than was CR4. However, there was some variation among AM phi isolates from different donors. In seven isolates, no CR4 modulation was produced with anti-CR4, whereas in six other isolates, CR4 was modulated by 66%. Incubation of cells in cytochalasin D increased modulation of both CR3 and CR4 on mAb-coated surfaces. Cells exhibiting increased mobility of CR4 showed an increased ability to form CR4-dependent EC3bi rosettes. The data are consistent with the hypothesis that CR3 and CR4 exhibit a variable association with the cytoskeleton that regulates their mobility and function. A relatively mobile subset of CR3 and/or CR4 mediates EC3bi attachment, whereas a relatively immobile subset of CR3 and/or CR4 fails to mediate EC3bi attachment but functions to promote ingestion of EC3bi.

Phorbol ester induces transient focal concentrations of functional, newly expressed CR3 in neutrophils at sites of specific granule exocytosis.

Treatment of neutrophils with phorbol myristate acetate (PMA) increases surface expression of CR3 (iC3b-receptor; CD11b/CD18). This up-regulation was examined in whole-mount preparations of adherent neutrophils by stereo high voltage immunoelectron microscopy. In the absence of PMA, immunogold-labeled CR3 was uniformly distributed in the plasma membrane. After 5 to 15-min incubations with PMA, when the highest rates of specific granule exocytosis occurred, the average density of CR3 in the membrane doubled. During this time, dense foci of CR3 were observed in addition to the uniform distribution of CR3. These dense concentrations of CR3 colocalized with secreted lactoferrin (LF), a specific granule marker, above assemblies of cytoplasmic vesicles that were morphologically similar to specific granules and contained LF. After longer incubations in PMA, LF secretion ceased, LF staining became rare, and the high density areas of CR3 were no longer present. These data demonstrate that incipient CR3 appear on the cell surface in high concentration at sites of specific granule exocytosis and then diffuse out into the plasmalemma. PMA also induced shedding of CR3 from the cell surface at the cell margins on structures which also contained LF. Shed CR3 was immunoprecipitated from incubation supernatants and shown to be of identical subunit composition to surface CR3. Although others have shown that the mobile, incipient surface CR3 do not mediate neutrophil adherence to endothelium or homotypic aggregation, the current experiments demonstrated that such CR3 do mediate iC3b-dependent adhesion. The rapid appearance and subsequent dissipation of high concentrations of CR3 on the neutrophil surface caused by specific granule fusion may be essential for neutrophil function at sites of complement deposition.

Analysis of the different types of leukocyte membrane complement receptors and their interaction with the complement system.

The specificity, distribution, and structure of 8 different types of leukocytes membrane complement (C) receptors (CR1, CR2, CR3, and receptors for C1q, beta 1H, C3e, C3a, and C5a) are discussed. Recent data are reviewed on the synthesis of C components by macrophages and B lymphocytes, and how these components may function in the activation of these two cell type by the C system. Commonly used C receptor assay procedures are evaluated in terms of both specificity and sensitivity. Specific assay procedures are recommended for measuring CR1 (C4b-C3b receptor), CR2 (C3d receptor), CR3 (C3bi receptor), and the beta 1H receptor. Assays include both rosette and fluorescence procedures for detection of C receptors on either mouse or human leukocytes. Primary systems have been selected for optimal sensitivity and specificity, and where possible, acceptable alternative systems that are less sensitive or specific are suggested for laboratories lacking facilities for C purification.