Complement Component C3bi Research Articles

Phagocytosis of optically-trapped particles: delivery of the pure phagocytic signal

852-854. doi:10.1038/sj.cr.7310105; published online 16 November 2006Phagocytosis is a fundamental cell biological process exhibited by a wide variety of cell types from single cell organ-isms, which rely on this for feeding, to phagocytes in higher animals, which rely on specialised immune cells for com-bating infecting micro-organisms. In the immune system, both macrophages and neutrophils play roles as phagocytes. Neutrophils are often called “professional phagocytes” because of their remarkable capacity for phagocytosis, being able to internalise microscopic particles (diam 0.5-3 mm) of virtually any surface material. The efficiency and speed of phagocytosis is, however, increased by coating the surface of the particles with opsonins such as antibodies or the complement component C3bi (acting on b2 integrin receptors), and C3bi-accelerated phagocytosis by neutrophils is the first line of defence by the innate immune system

Cytosolic free Ca(2+) changes and calpain activation are required for beta integrin-accelerated phagocytosis by human neutrophils.

Phagocytosis of microbes coated with opsonins such as the complement component C3bi is the key activity of neutrophils. However, the mechanism by which opsonins enhance the rate of phagocytosis by these cells is unknown and has been difficult to study, partly because of the problem of observing and quantifying the events associated with phagocytosis. In this study, C3bi-opsonized particles were presented to neutrophils with a micromanipulator, so that the events of binding, pseudopod cup formation, engulfment, and completion of phagocytosis were clearly defined and distinguished from those involved with chemotaxis. Using this approach in combination with simultaneous phase contrast and Ca2+ imaging, the temporal relationship between changes in cytosolic free Ca2+ concentration and phagocytosis were correlated. Here we show that whereas small, localized Ca2+ changes occur at the site of particle attachment and cup formation as a result of store release, rapid engulfment of the particle required a global change in cytosolic free Ca2+ which resulted from Ca2+ influx. This latter rise in cytosolic free Ca2+ concentration also liberated a fraction of β2 integrin receptors which were initially immobile on the neutrophil surface, as demonstrable by both fluorescence recovery after laser bleaching and by visualization of localized β2 integrin labelling. Inhibitors of calpain activation prevented both the Ca2+-induced liberation of β2 integrin and the rapid stage of phagocytosis, despite the persistence of the global Ca2+ signal. Therefore, we propose that Ca2+ activation of calpain causes β2 integrin liberation, and that this signal plays a key role in the acceleration of β2 integrin–mediated phagocytosis.

Dynamics of cytoskeletal proteins during Fcgamma receptor-mediated phagocytosis in macrophages.

Particle ingestion by phagocytosis results from sequential rearrangements of the actin cytoskeleton and overlying membrane. To assemble a chronology of molecular events during phagosome formation and to examine the contributions of phosphoinositide 3-kinase (PI 3-kinase) to these dynamics, a method was developed for synchronizing Fcgamma receptor-mediated phagocytosis by murine macrophages. Erythrocytes opsonized with complement component C3bi were bound to macrophages at 37degrees C, a condition that does not favor particle phagocytosis. Addition of soluble anti-erythrocyte IgG resulted in rapid opsonization of the bound erythrocytes, followed by their immediate internalization via phagocytosis. Cellular content of F-actin, as measured by binding of rhodamine-phalloidin, increased transiently during phagocytosis, and this increase was not diminished by inhibitors of PI 3-kinase. Immunofluorescence localization of myosins in macrophages fixed at various times during phagocytosis indicated that myosins II and IXb were concentrated in early phagosomes, myosin IC increased later, and myosin V appeared after phagosome closure. Other cytoskeletal proteins showed similar variations in the timing of their appearance in phagosomes. The PI 3-kinase inhibitor wortmannin did not change the dynamics of PI 3-kinase or ezrin localization but prevented the loss of PAK1 from phagosomes. These results suggest that PI 3-kinase deactivates PAK1, and that this may be needed for phagosome closure.

Granulocyte activation induced by intense interval running.

Activation of granulocytes has been associated with normal immune function, inflammation, and exercise-induced muscle damage. The effect of intense interval running on granulocyte activation was examined by use of flow cytometry, monoclonal antibodies, and spectrophotometric techniques. Eight trained males [maximal oxygen uptake VO2max, mean (SD) = 64.4 (3.6) ml/kg/min; age 30.1 (4.8) years] undertook an intense interval exercise (treadmill running) protocol to exhaustion. Subjects completed an average of 16.5 one-minute runs. Granulocyte expression of CR3 (CD11b), receptor for complement component C3bi (6 and 24 h post-test), and Fc gamma RIII (CD16) (24 h post-test) and the plasma concentration of elastase-inhibitor complex (1 h post-test) increased significantly (all P < .05). Subjects (8 of 8) exhibited a post-test decrease at either 1 or 6 h (P < .01) and a 24-h post-test significant increase (7 of 8; P < .05) in granulocyte 90 degrees light scattering (LS). Plasma lactoferrin (Lf) concentration, although increased by 17% at 6 h post-test, was not significantly different from resting values at any sampling point. Changes in plasma Lf and median channel 90 degrees LS were significantly correlated (r = -.43, P = .04), raising the possibility of monitoring exercise-induced granulocyte activation (degranulation) by flow cytometry. Intense interval exercise appears to induce granulocyte activation, as manifested by release of granule proteins and changes in 90 degrees LS and expression of both Fc and complement receptors.

Adhesion-promoting receptors on leukocytes

Leukocytes express a family of cell-surface glycoproteins, CDllaKD18 (IRA-l), CDllb/ CD18 (CR3), and CDllKD18 (p150,95), that perform a broad range of adhesive functions. As detailed below, this family of receptors mediates the binding of cytolytic T cells to targets, the interaction of phagocytes with microbes opsonized with complement component C3bi, and the adherence of leukocytes to the vascular endothelium prior to diapedesis. Although the CDllXD18 family is restricted to leukocytes, recent work shows that these receptors belong to a large, broadly distributed fanily of adhesion-promoting receptors called integrins (Hynes, Cell 1987, 48:549-554). This review will focus on recent studies concerning the structure and function of the CDllKD18 family of receptors.

Roles of CR3 and mannose receptors in the attachment and ingestion of Leishmania donovani by human mononuclear phagocytes.

Leishmania donovani is an obligate intracellular parasite of mammalian macrophages. Two macrophage receptors, the mannose-fucose receptor (MFR) and the receptor for complement component C3bi, CR3, were examined for their roles in the attachment and ingestion of L. donovani by human monocyte-derived macrophages. Two monoclonal antibodies which bind to the human CR3, anti-Mo1 and anti-Mac-1, inhibited both attachment and ingestion of L. donovani promastigotes after preincubation with human monocyte-derived macrophages; attachment was inhibited by 40 and 62% by anti-Mo1 and anti-Mac-1, respectively, and ingestion was inhibited by 34 and 51% by anti-Mo1 and anti-Mac-1, respectively. The interaction between promastigotes and CR3 may not have involved the C3bi-binding site on CR3, however, because a monoclonal antibody which exhibits specificity for this site, OKM10, inhibited promastigote attachment by only 18%. In contrast, OKM1, which is believed to react with the alternate lectinlike binding site on CR3, inhibited ingestion by 65%. MFR activity was inhibited using the soluble MFR ligands, mannan and mannosylated bovine serum albumin, which also inhibited promastigote attachment by 40 and 37%, respectively. The simultaneous inhibition of both CR3 (by anti-Mac-1) and the MFR (by either mannan or mannosylated bovine serum albumin) resulted in a greater decrease in promastigote attachment than inhibition of either receptor alone. Additionally, the reduction of MFR activity by allowing macrophages to adhere to a mannan-coated surface followed by the addition of anti-CR3 antibodies resulted in an 81% inhibition of promastigote ingestion, a greater decrease than was obtained by manipulation of either receptor alone. The results suggest that the MFR and CR3 independently participate in the attachment and ingestion of L. donovani promastigotes by human macrophages.

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.

A genomic clone encoding the alpha chain of the OKM1, LFA-1, and platelet glycoprotein IIb-IIIa molecules.

LFA-1, an antigen involved in cytolytic T lymphocyte-mediated killing, and Mac-1, the receptor for complement component C3bi, constitute a family of structurally and functionally related cell surface glycoproteins involved in cellular interactions. In both mouse and man, Mac-1 (OKM1) and LFA-1 share a common 95-kDa beta subunit but are distinguished by their alpha chains, which have different cellular distributions, apparent molecular masses (165 and 177 kDa, respectively), and peptide maps. We report the isolation of a genomic clone from a human genomic library that on transfection into mouse fibroblasts produced a molecule(s) reactive with monoclonal antibodies to OKM1, to LFA-1, and to platelet glycoprotein IIb-IIIa. This gene was cloned by several cycles of transfection of L cells with a human genomic library cloned in lambda phage Charon 4A and subsequent "rescue" of the lambda phage. Transfection with the purified recombinant lambda DNA yielded a transfectant that expressed the three human alpha chains of OKM1, LFA-1, and glycoprotein IIb-IIIa, presumably in association with the murine beta chain.

Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor.

Recombinant human tumor necrosis factor (TNF) was found to enhance the adherence of human peripheral blood neutrophils to human umbilical vein endothelial (HUVE) cell monolayers in vitro. The enhancement was due to the effects both on neutrophils and HUVE cells. The effect on neutrophils was maximally induced within 5 min and did not require protein or RNA synthesis. By contrast, maximal effects on HUVE cells took 4 hr to develop and required de novo protein and RNA synthesis; however, exposure of HUVE cells to TNF for as little as 5 min was sufficient to initiate changes leading to maximal adherence of neutrophils at 4 hr. Both the effect on neutrophils and that on HUVE cells were blocked by a monoclonal antibody against TNF. TNF also rapidly induced an increased surface expression of neutrophil antigens recognized by monoclonal antibodies directed against epitopes of a glycoprotein required for optimum adherence and for complement component C3bi receptor (CR3) function. Thus, the mechanism of action of TNF may involve the regulation of expression of cell surface molecules. Our observations show that TNF induces a process central to the development of all inflammatory reactions and that both blood neutrophils and endothelial cells are targets of TNF action. The regulation of inflammatory reactions by TNF or antagonists of TNF has wide-ranging clinical implications.