C3bi Receptor Research Articles

A neutrophil membrane marker reveals two groups of chronic myelogenous leukemia and its absence may be a marker of disease progression

An IgG1 monoclonal antibody, 31D8, that recognizes normal neutrophil (PMN) membranes, was used to study PMN from patients with chronic myelogenous leukemia (CML). Nineteen patients with Philadelphia chromosome positive CML were followed over a ten-month period and compared with 23 normals, six patients with leukemoid reactions, and eight patients with phagocytic cell defects. The percentage of PMN binding of 31D8 among normal subjects was variable about a normal distribution with an average of 95 +/- 2% of cells binding 31D8. In contrast, there were two groups of CML patients: in 14 patients 88 +/- 3% PMN bound 31D8 while in the remaining five patients only 6 +/- 6% PMN bound 31D8. PMN 31D8 binding was normal in the control patient groups. Control antibodies 7C3 (binds to PMN precursors) and OKM1 (binds to the CR3 (iC3b) receptor) bound normally to CML neutrophils. Functionally, CML cells had normal chemotaxis to several stimuli and normal superoxide generation to phorbol myristate acetate. However, superoxide production in response to fmet-leu-phe was significantly less in 31D8 negative CML PMN than both 31D8 positive CML PMN and normal PMN which contained 85% 31D8 positive and 15% 31D8 negative PMN. Clinically, 2 of 14 CML patients with 31D8 positive PMN were in blast crisis (one extramedullary) at the time of study and the other 12 patients remained clinically stable in the chronic phase during the ten months of study. In contrast, one of five patients with 31D8 negative PMN was in blast crisis at the time of study and all four of the remaining patients progressed to either the accelerated phase or blast crisis. Three of these patients died of their disease eight to ten months after their initial study. Thus, failure of CML cells to bind 31D8 may be useful for predicting which patients are likely to progress to the accelerated phase or blast crisis.

A Neutrophil Membrane Marker Reveals Two Groups of Chronic Myelogenous Leukemia and Its Absence May Be a Marker of Disease Progression

An IgG1 monoclonal antibody, 31D8, that recognizes normal neutrophil (PMN) membranes, was used to study PMN from patients with chronic myelogenous leukemia (CML). Nineteen patients with Philadelphia chromosome positive CML were followed over a ten-month period and compared with 23 normals, six patients with leukemoid reactions, and eight patients with phagocytic cell defects. The percentage of PMN binding of 31D8 among normal subjects was variable about a normal distribution with an average of 95 +/- 2% of cells binding 31D8. In contrast, there were two groups of CML patients: in 14 patients 88 +/- 3% PMN bound 31D8 while in the remaining five patients only 6 +/- 6% PMN bound 31D8. PMN 31D8 binding was normal in the control patient groups. Control antibodies 7C3 (binds to PMN precursors) and OKM1 (binds to the CR3 (iC3b) receptor) bound normally to CML neutrophils. Functionally, CML cells had normal chemotaxis to several stimuli and normal superoxide generation to phorbol myristate acetate. However, superoxide production in response to fmet-leu-phe was significantly less in 31D8 negative CML PMN than both 31D8 positive CML PMN and normal PMN which contained 85% 31D8 positive and 15% 31D8 negative PMN. Clinically, 2 of 14 CML patients with 31D8 positive PMN were in blast crisis (one extramedullary) at the time of study and the other 12 patients remained clinically stable in the chronic phase during the ten months of study. In contrast, one of five patients with 31D8 negative PMN was in blast crisis at the time of study and all four of the remaining patients progressed to either the accelerated phase or blast crisis. Three of these patients died of their disease eight to ten months after their initial study. Thus, failure of CML cells to bind 31D8 may be useful for predicting which patients are likely to progress to the accelerated phase or blast crisis.

Complement, membrane glycoproteins, and complement receptors: Their role in regulation of the immune response

To determine the effect of complement on the normal antibody response we studied seven patients with genetically determined complement component deficiencies, guinea pigs deficient of C4 and C2, respectively, and two patients whose neutrophils and monocytes lack the C3bi receptor. Patients deficient of early complement components (C4, C2, C3) have abnormal antibody responses to the T-cell-dependent antigen, bacteriophage φX 174. Complement-deficient guinea pigs (C4, C2) produce less antibody than normal guinea pigs and are unable to maintain measurable antibody levels; during secondary immunization they fail to develop amplification and to switch from IgM to IgG. This defect can be overcome by increasing the antigen dose or by injecting normal guinea pig serum at the time of the primary (but not the secondary) immunization. Patients with deficiency of the C3bi receptor were shown to have a significantly depressed antibody response to T-dependent antigens. We postulate that the contribution of complement to the mature humoral immune response is related to activation of C3. The initial production of IgM following antigen injection leads to antigen-antibody complexes which interact with complement, to be nonspecifically trapped by C3b and C3bi receptors on B cells or macrophages. Thus antigen is selectively accumulated within the lymphoid organs and in turn may entrap antigen-specific B cells by interaction of the trapped antigen with surface immunoglobulin. As a result, close approximation between antigen, antibody, and a network of specific and nonspecific lymphoid cells is initiated, allowing generation of specific memory cells and initiation of a prompt mature antibody response on subsequent exposure to antigen.

A monoclonal antibody (3A33) that reacts with a mouse‐specific epitope of Mac‐1 antigen

The 3A33 monoclonal antibody, obtained by fusing rat immune lymphocytes with mouse plasmacytoma cells, was directed against mouse macrophages. Antibody 3A33, a rat IgG2a, reacted with macrophages from all the mouse strains tested, with mouse blood monocytes and with 56% of bone marrow cells, but not with T lymphocytes. It immunoprecipitated an antigen with alpha and beta subunits, found to be identical to Mac-1 antigen after cross-absorption experiments with M1/70 monoclonal antibody. The two antigenic determinants of the Mac-1 molecule identified by the 3A33 and M1/70 antibodies both displayed reduced expression on inflammatory macrophages and comparable resistance to trypsin digestion. The sites of the determinants on this molecule seemed close together judging from the ability of both the 3A33 and M1/70 antibodies to block C3bi receptor sites and compete for cell binding. However, unlike antibody M1/70, 3A33 never reacted with human cells bearing Mac-1 antigen. Therefore, two closely related epitopes of the Mac-1 molecule - one specific for mouse and one common to mouse and man, were recognized by these monoclonal antibodies.

Phagocyte defects

Although inherited forms of phagocyte defects affect a small proportion of the general population, their clinical course can be altered dramatically by a physician's awareness of these diseases and modifications of the approach to and treatment of affected patients. The most common syndromes are chronic granulomatous disease of childhood (CGD), the Chediak-Higashi syndrome (CHS), the hyperimmunoglobulin-E-recurrent infection (Job's) syndrome (HIE), and myeloperoxidase (MPO) deficiency. CGD patients have defects in the oxidative metabolism involved in killing catalase-positive organisms. CHS patients have giant granules defective in fusing with phagosomes and subsequent killing of ingested organisms. HIE patients have abnormal chemotaxis and elevated IgE levels and are susceptible to skin infections with Staphylococcus aureus and recurrent sinopulmonary infections. MPO-deficient patients often go undetected since they rarely have recurrent infections unless they have a concomitant disease such as diabetes mellitus. Patients with a recently described syndrome, C3bi receptor deficiency, have recurrent bacterial infections and persistent leukocytosis, and their neutrophils have abnormal adherence and phagocytosis. The absence of specific granules is a more rare entity but these patients also have recurrent infections thought to be secondary to a chemotactic defect and a minor abnormality of microbial killing exhibited by their neutrophils. This review will focus on the clinical presentation and management of these patients.

Inherited deficiencies of complement and complement-related proteins

The complement cascade and cell-surface proteins related to the complement system are critically important to host defense, immune complex catabolism, and possibly immunoregulation. Genetically determined deficiency states have been described for many complement proteins as well as for fluid phase and cell-borne inhibitors of the complement cascade and cellular complement receptors. Autoimmune diseases and enhanced susceptibility to infection are the dominant clinical manifestation of these deficiencies. Classical pathway deficiencies and low numeric expression of erythrocyte C3b receptors (CR1) are typically associated with autoimmune disorders, while aternative pathway, terminal component, and leukocyte iC3b receptor (CR3) defects predispose to pyogenic bacterial disease. This distinction is not absolute, however, and both classes of disease may appear in most of the reported deficiency states. Specific pharmacologic therapy exists for C1-inhibitor deficiency (hereditary angioedema). Management of other complement deficiencies currently involves sustained diagnostic suspicion and meticulous management of complicating diseases.

Comparison of intravenous gamma globulin and a monoclonal anti-Fc receptor antibody as inhibitors of immune clearance in vivo in mice.

Fc-receptor-mediated clearance and nonspecific phagocytic clearance were assessed after the infusion of monomeric human IgG, heat-aggregated human IgG, and a monoclonal anti-mouse macrophage FcII receptor antibody (2.4G2) into normal mice. Each agent blocked Fc-receptor function in vivo, but 2.4G2 was much more potent per microgram than the other agents. Monomeric IgG in blocking doses did not affect other aspects of immune function. In contrast, aggregated IgG, and to a lesser extent, 2.4G2 reduced serum complement levels. In addition, these agents also caused moderate reductions in nonspecific phagocytic function. Monoclonal anti-mouse macrophage C3bi receptor antibody (Mac-1), another monoclonal antibody which binds to macrophage CR3 receptors without interfering with Fc-receptor function, also reduced serum complement and inhibited nonspecific phagocytic function. Complement depletion alone (produced by infusion of cobra venom factor) could not account for the observed changes in Fc receptor or nonspecific phagocytic function. We conclude that both monomeric IgG and anti-Fc-receptor antibodies can markedly inhibit Fc-receptor function in vivo; however, the pattern of physiologic changes produced by these agents differs.

Interferon-gamma depresses binding of ligand by C3b and C3bi receptors on cultured human monocytes, an effect reversed by fibronectin.

Cultivation of human monocytes with recombinant IFN-gamma causes a 5-10-fold depression in their binding of EC3b or EC3bi. This effect is observed within 18 h and is expressed for 5 d in the presence of 100 U/ml IFN-gamma. The capacity of IFN-gamma-treated phagocytes to bind EC3b and EC3bi is fully restored if the phagocytes are allowed to spread for 45 min on surfaces coated with Fn. IFN-gamma-treated cells express normal levels of cell surface C3b and C3bi receptors as measured with monoclonal anti-receptor antibodies, and spreading on Fn does not alter receptor number. We conclude that cultivation with IFN-gamma causes a change in the nature of these receptors that prevents them from interacting with ligand. Immunoelectron microscopy shows that C3bi receptors are expressed on the apical surface of the IFN-gamma-treated MO and that these receptors exhibit normal capacity to migrate in the plane of the membrane. Thus, the nature of the change caused by IFN-gamma is not related to changes in receptor number, location, or mobility. While spreading of IFN-gamma-treated cells on Fn enables C3 receptors to bind ligand, it does not enable them to promote phagocytosis. Treatment of cells with PMA alone does not affect binding or phagocytosis, but treatment of cells with both Fn and PMA enables cells to phagocytose EC3b and EC3bi. These data indicate that the binding and signaling activities of C3 receptors are separately regulated. Fn enables receptors to bind ligand and PMA enables them to signal phagocytosis.

Murine eosinophil differentiation factor. An eosinophil-specific colony-stimulating factor with activity for human cells.

A purified murine lymphokine, eosinophil differentiation factor (EDF), was found to be a selective stimulus for the clonal proliferation and differentiation of murine eosinophil progenitor cells, establishing it as the murine eosinophil colony-stimulating factor (Eo-CSF). EDF was also active on human eosinophil progenitors and mature blood eosinophils, but had no effect on neutrophil or macrophage precursor cells, nor on blood neutrophils. In culture of human bone marrow cells, EDF stimulated equal numbers and equal sizes of eosinophil colonies to develop when compared with human placental conditioned medium, a source of human CSFs, suggesting that all responsive progenitor cells were stimulated. Clone transfer experiments and the linear relationship between number of bone marrow cells plated and colonies produced confirmed that the action of EDF was directly on eosinophil progenitor cells. EDF increased the capacity of human blood eosinophils, but not neutrophils, to kill antibody-coated tumor cells and to phagocytose serum-opsonized yeast cells. This functional activation was associated with the enhanced expression of functional antigens (GFA-1, GFA-2, and the receptor for C3bi) on eosinophils. The possession by EDF (Eo-CSF) of all the properties expected of a human eosinophil CSF raises the possibility that a human analog of this molecule exists, and is involved in the regulation of production and function of human eosinophils in vivo.

Neutrophil activation detected by monoclonal antibodies.

Monoclonal antibodies have been produced against three neutrophil-associated membrane proteins (p 90, p 170, and p 70) expressed at different maturation stages of the cells. The reactivity of the antibodies against p 90 (B13.9) and p 170 (CLB-gran 10), as measured by quantitative flow cytofluorometry, increased after stimulation of the neutrophils by the calcium ionophore A23187, by phorbol myristate acetate, or by the chemoattractant formylmethionyl-leucyl-phenylalanine in combination with cytochalasin B. This increase is regulated independently of the simultaneously increased expression of the C3bi receptor, because neutrophils of a patient deficient for the C3bi receptor showed a normal increase in membrane expression of p 90 and p 170. Neutrophil cytoplasts were not inducible to increased membrane expression, suggesting that the cytoplasts lack the internal pool of these proteins. The reactivity of the antibody against p 70 (CLB-gram 5) was not affected by activation. The antibodies B13.9 and CLB-gran 10 may be useful to detect neutrophil activation.

Effect of human serum and some of its components on neutrophil adherence and migration across an epithelium.

The effect of human serum and some of its components on the process of transepithelial migration of human neutrophils was investigated in an in vitro system. 10% autologous serum caused an increase in neutrophil adherence to and migration across canine kidney epithelial cells. This increase in neutrophil binding also occurred if the epithelium but not the neutrophils had been preincubated with serum. The binding was lost if the serum was either preabsorbed over the kidney epithelium before use or heat inactivated. Indirect immunofluorescence studies indicated that IgG, IgM, and a component of C3 bound to the epithelial surface, whereas IgA, IgE, or C5a were not detectable. The majority of epithelial cells were immunofluorescent, however epithelial cells with varying degrees of reactivity were also apparent and approximately 5% of the epithelial cells did not bind IgG, IgM, and C3. When epithelia were simultaneously tested for the presence of either IgG, IgM, or C3, and bound neutrophils the few epithelial cells which did not bind IgG or IgM also did not bind C3 or neutrophils. Studies with monoclonal antibodies against Fc and C3 receptors indicate that neutrophil adherence to the epithelial surface was mediated predominately by the receptors for C3b and C3bi. In response to a chemotactic gradient, bound neutrophils were able to detach and migrate across the epithelium. A separate heat-stable factor(s) in serum was able to increase neutrophil migration across the epithelial monolayer. This factor acted independently of the factors which caused the increase in neutrophil binding as the increase in neutrophil migration also occurred under conditions (preabsorption over the kidney epithelium or heat inactivation) that prevented the increase in neutrophil binding. The increase in neutrophil migration may be caused by the permeability-increasing properties of this factor as both serum and heat-inactivated serum lowered the transepithelial electrical resistance an average of 38 and 35%, respectively, in 40 min. Upon removal of serum or heat-inactivated serum, the resistance returned 100 and 81%, respectively, in 5 h.

Activation of Human Neutrophils by Monoclonal Antibody PMN7C3: Cell Movement and Adhesion Can Be Triggered Independently From the Respiratory Burst

Anti-neutrophil monoclonal antibody PMN7C3 (IgG3) recognizes glycoproteins bearing the oligosaccharide lacto-N-fucopentaose III, including the C3bi receptor, LFA-1, and p150,95 on the plasma membrane and a group of granule-associated glycoproteins. We have previously shown that binding of this antibody to polymorphonuclear leukocytes (PMNs) stimulates a transient rise in cytosolic free calcium concentration but does not trigger the neutrophil respiratory burst. We now demonstrate that binding of PMN7C3 (and five other monoclonal antibodies recognizing the same antigen) to human neutrophils activates several other cellular responses. Addition of PMN7C3 to monolayers of neutrophils induces a rapid change in cell shape followed by pseudopod formation and increased migration. With incubation at 37 degrees C, the neutrophils aggregate in clusters (leukoagglutination). Quantitation of cell movement in a multiwell chemotaxis assembly or by migration of PMNs under agarose revealed that PMN7C3 is both chemotactic and chemokinetic. Pretreatment with the antibody inhibits subsequent chemotactic response to other stimuli. Monoclonal antibodies binding to other neutrophil antigens do not mimic these effects. These data suggest that cell movement and adhesion can be triggered independently from the respiratory burst. PMN7C3 may be a useful probe with which to study the events that link receptor-ligand binding to cellular response.

Metabolic comparison between basophils and other leukocytes from human blood.

Basophilic granulocytes were purified from the blood of normal individuals by successive isopyknic centrifugation and elutriation centrifugation. Starting with the leukocyte-rich fraction of 500 ml of blood, we recovered 31 to 80% (mean 51%, n = 20) of the basophils in 45 to 87% purity (mean 69%, n = 23). The contaminating cells were mainly lymphocytes. The basophils were greater than 98% vital (exclusion of ethidium bromide and hydrolysis of fluorescein diacetate). The histamine content of the basophils was 1.1 to 2 pg/cell (mean 1.6 pg/cell, n = 22). With anti-IgE, 30 to 50% of the histamine was released; with phorbol myristic acetate (PMA) or the calcium ionophore A23187, 70 to 100% of the histamine was released. Serum-opsonized zymosan (STZ) did not induce histamine release. Reactions with monoclonal antibodies revealed that the basophils expressed the C3bi receptor (CR3) and the leukocyte function-associated antigen 1 (LFA1), but not the gp 150,95 antigen, the C3b receptor (CR1), or the low avidity Fc gamma receptor. Basophils carry class I but not class II HLA antigens. During incubation of the basophils with serum-opsonized Staphylococcus aureus or Escherichia coli, these bacteria were neither phagocytized nor killed. STZ, PMA, A23187, or anti-IgE did not initiate an "oxidative burst" in the basophils. This was tested with oxygen consumption, cytochrome c reduction, NBT reduction, chemiluminescence, and release of hydrogen peroxide. Moreover, we did not detect cytochrome b558, superoxide dismutase, catalase, or peroxidase in the basophils. Of the typical granule-associated enzymes lysozyme, Vitamin B12-binding protein, and beta-glucuronidase, only beta-glucuronidase was present in the basophils in detectable amounts. This enzyme was released, together with histamine, on incubation of the cells with PMA, A23187, or anti-IgE, but not with STZ. We conclude that basophils from normal human blood are not phagocytes and are probably not involved in the oxidative defense of the host against foreign antigens.

Activation of human neutrophils by monoclonal antibody PMN7C3: cell movement and adhesion can be triggered independently from the respiratory burst

Anti-neutrophil monoclonal antibody PMN7C3 (IgG3) recognizes glycoproteins bearing the oligosaccharide lacto-N-fucopentaose III, including the C3bi receptor, LFA-1, and p150,95 on the plasma membrane and a group of granule-associated glycoproteins. We have previously shown that binding of this antibody to polymorphonuclear leukocytes (PMNs) stimulates a transient rise in cytosolic free calcium concentration but does not trigger the neutrophil respiratory burst. We now demonstrate that binding of PMN7C3 (and five other monoclonal antibodies recognizing the same antigen) to human neutrophils activates several other cellular responses. Addition of PMN7C3 to monolayers of neutrophils induces a rapid change in cell shape followed by pseudopod formation and increased migration. With incubation at 37 degrees C, the neutrophils aggregate in clusters (leukoagglutination). Quantitation of cell movement in a multiwell chemotaxis assembly or by migration of PMNs under agarose revealed that PMN7C3 is both chemotactic and chemokinetic. Pretreatment with the antibody inhibits subsequent chemotactic response to other stimuli. Monoclonal antibodies binding to other neutrophil antigens do not mimic these effects. These data suggest that cell movement and adhesion can be triggered independently from the respiratory burst. PMN7C3 may be a useful probe with which to study the events that link receptor-ligand binding to cellular response.

Monoclonal Antibodies Binding to the Human Neutrophil C3bi Receptor Have Disparate Functional Effects

Abstract Three monoclonal antibodies (MAb)--OKMI, 7C3, and 60.3-- immunoprecipitated a common 170-kd neutrophil membrane antigen closely associated with, or identical to, the C3bi receptor (CR3). Despite binding to a common receptor, these antibodies displayed marked differences in their effects on C3bi-mediated neutrophil function as assessed by the binding and ingestion of opsonized zymosan and the subsequent triggering of the respiratory burst. Antibody 7C3 caused a time-dependent, irreversible inhibition of the neutrophil oxidative response to opsonized zymosan that correlated with capping of the bound antibody. In contrast, antibody 60.3 caused an immediate inhibition of the neutrophil oxidative response to opsonized zymosan that required the continuous presence of exogenous antibody to achieve the maximal inhibitory effect. Antibody OKMI demonstrated minimal inhibition of O2- release. Despite their functional differences, binding of either 7C3 or 60.3 led to up-regulation of new antigen, presumably from intracellular sites as previously described using OKMI. Crossed immunoprecipitations of radiolabeled neutrophil lysates indicated that each MAb bound to different antigens near or within the CR3 complex. Thus three MAb binding to the neutrophil CR3 receptor each caused receptor up- regulation but had markedly different functional effects on the cell.

Monoclonal antibodies binding to the human neutrophil C3bi receptor have disparate functional effects

Three monoclonal antibodies (MAb)--OKMI, 7C3, and 60.3--immunoprecipitated a common 170-kd neutrophil membrane antigen closely associated with, or identical to, the C3bi receptor (CR3). Despite binding to a common receptor, these antibodies displayed marked differences in their effects on C3bi-mediated neutrophil function as assessed by the binding and ingestion of opsonized zymosan and the subsequent triggering of the respiratory burst. Antibody 7C3 caused a time-dependent, irreversible inhibition of the neutrophil oxidative response to opsonized zymosan that correlated with capping of the bound antibody. In contrast, antibody 60.3 caused an immediate inhibition of the neutrophil oxidative response to opsonized zymosan that required the continuous presence of exogenous antibody to achieve the maximal inhibitory effect. Antibody OKMI demonstrated minimal inhibition of O2- release. Despite their functional differences, binding of either 7C3 or 60.3 led to up-regulation of new antigen, presumably from intracellular sites as previously described using OKMI. Crossed immunoprecipitations of radiolabeled neutrophil lysates indicated that each MAb bound to different antigens near or within the CR3 complex. Thus three MAb binding to the neutrophil CR3 receptor each caused receptor up-regulation but had markedly different functional effects on the cell.

Antigen-specific functions of a CD4+ subset of human T lymphocytes with granular morphology.

We previously showed that a small proportion of the CD4+ human lymphocytes express the C3bi receptor (CD11). In the present investigation these cells were found to have the homogeneous morphology of granular lymphocytes. Between 70 and 80% of the cells reacted with antibody Leu-7, but they did not express Fc receptors and had no spontaneous cytotoxic activity against the NK-sensitive cell line K562. The CD11+/CD4+ cells uniformly expressed CD3 and could be induced to express IL 2 receptors by activation with PHA or anti-CD3 antibody. Compared with unfractionated T cells, however, CD11+/CD4+ granular lymphocytes had a reduced ability to produce IL 2 after stimulation with PHA or anti-CD3 antibody. Nevertheless, the CD11+/CD4+ subset generated specific allocytotoxicity after stimulation with allogeneic cells and culture in IL 2. Furthermore, the CD11+/CD4+ cells also proliferated after stimulation with tetanus toxoid and PPD. Thus some cells of the CD11+/CD4+ subset are capable of antigen-specific responses. These results indicate that certain CD11+ cells with granular morphology are mature T lymphocytes.

Exudation primes human and guinea pig neutrophils for subsequent responsiveness to the chemotactic peptide N-formylmethionylleucylphenylalanine and increases complement component C3bi receptor expression.

After circulating in the vascular system a short time, polymorphonuclear leukocytes (PMN) migrate to extravascular sites in response to chemotactic stimuli. Prestimulation of PMN in vitro by secretagogues has been shown to increase their number of N-formylmethionylleucylphenylalanine (fmet-leu-phe) and complement component C3bi (CR3) receptors. We investigated whether the same phenomenon occurred in vivo, comparing characteristics of human skin chamber and guinea pig peritoneal exudate and blood PMN. Exudate PMN of both species contained approximately 28% less of the specific granule marker vitamin B12-binding protein (P less than 0.01) but a similar amount of the azurophil granule marker beta-glucuronidase. The total number of fmet-leu-phe receptors was 5.9 times higher in guinea pig exudate than in blood PMN (P less than 0.01) and 2.9 times higher in human exudate than in blood PMN (P less than 0.02). All exudate PMN and most blood PMN preparations showed a high affinity receptor (Kd approximately 2.3 X 10(-8) M) and a low affinity receptor (approximately 1.5 X 10(-7) M). The upregulation of fmet-leu-phe receptors in exudate PMN correlated with an improved responsiveness to fmet-leu-phe induced membrane depolarization, oxidative metabolism, and chemotaxis. In addition, the concentration of fmet-leu-phe that produced a half-maximal response of chemotaxis, superoxide production, and membrane potential depolarization was 10-fold lower in exudate PMN than in blood PMN. Human exudate PMN had a twofold increased C3bi receptor expression compared with blood PMN. Thus, a preferential loss of specific granules is associated with increased number of high and low affinity fmet-leu-phe receptors and increased C3bi receptor expression not only in vitro, but also in vivo. The data indicate that exudation primes PMN for their subsequent responsiveness to fmet-leu-phe, a modification that may be crucial for efficient antimicrobial host defense.

Phorbol esters cause sequential activation and deactivation of complement receptors on polymorphonuclear leukocytes.

Phorbol myristate acetate (PMA) exerts a biphasic effect on receptors for C3b and C3bi of human polymorphonuclear leukocytes (PMN). The addition of PMA for 10 min enhances the capacity of these receptors to promote binding and phagocytosis of C3b- and C3bi-coated erythrocytes. Upon additional incubation for 60 min, the capacity of these receptors to bind and ingest ligand-coated erythrocytes decreases to levels below those of resting cells. Although PMA does cause increased expression of cell surface C3b and C3bi receptors, the sequential rise and fall of receptor activity cannot be accounted for by alterations in the number of surface receptors. It appears, rather, that PMA causes qualitative changes in these receptors, first an increase in receptor activity (activation) and then a decrease in receptor activity (deactivation). In contrast with its effects on C3 receptors, PMA causes only a reduction in the capacity of Fc receptors to bind and ingest IgG-coated erythrocytes. Deactivation of Fc receptors also appears to involve a qualitative alteration in receptors, because the binding affinity of soluble immune complexes is sharply reduced upon stimulation of PMN with PMA. To explore the role of phosphorylation in the sequential activation and deactivation of phagocytosis-promoting receptors, we loaded PMN with thiophosphate (thioP). This compound is incorporated into cellular nucleotides and proteins, and the resultant (thio)phosphorylated proteins are resistant to phosphatases. thioP-loaded cells show enhanced receptor activity, suggesting that activation of receptors is mediated by a phosphorylation event. Cells loaded with thioP and treated with PMA for 70 min do not deactivate C3 or Fc receptors, suggesting that the deactivation is the result of a dephosphorylation event.

Binding and catabolism of aggregated immunoglobulins containing C3b by U937 cells.

We studied Fc receptor and C3b receptor (CR1) function on U937 cells, a human monocyte cell line. C3b was incorporated into stable soluble heat aggregates of 125I-IgM (A-IgM) and 125I-IgG (A-IgG) by using functionally pure classical pathway components. C3b incorporation was verified by the ability of aggregates to bind to human red cells and by cosedimentation of 125I and 131I during ultracentrifugation. Cell uptake and degradation of A-IgG X C3b was increased up to twofold compared with A-IgG not containing C3b molecules. However, A-IgG X C3b bound to CR1 after Fc receptors were blocked with nonradiolabeled A-IgG were also not endocytosed and catabolized. Moreover, A-IgM X C3b was bound but not degraded by U937 cells. As expected, uptake of A-IgM without C3b was negligible. CR1-mediated binding of A-IgM X C3b was specifically inhibited both by a murine monoclonal antibody against the human CR1 that blocks C3b binding and by C3b oligomers generated by trypsin activation of C3, but not by monoclonal antibodies against the iC3b receptor (CR3). We conclude that CR1 on U937 cells cause increased binding of A-IgG, and this increased binding leads to increased Fc-mediated endocytosis and catabolism of model immune complexes. However, binding of soluble ligands by CR1 alone, even when binding is multivalent, does not lead to endocytosis and degradation of soluble ligands bearing C3b.