Participation of CR1 (CD35), CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in membranoproliferative glomerulonephritis type I.

Intraglomerular expression of complement receptors (CR) was investigated chronologically in 22 repeatedly biopsied patients with membranoproliferative glomerulonephritis (MPGN) type I by indirect immunoperoxidase staining using MoAbs. Patients were divided into two groups based on whether intraglomerular C3c deposition was decreased at the second biopsy (2nd Bx) (group A, n = 12), or not (group B, n = 10). At the first biopsy (1st Bx), the severity of glomerular injury and the degree of glomerular C3c deposition were compatible between the two groups. Four patterns of CR1 (CD35) expression on podocytes were recognized: normal; generally decreased; focally/segmentally lost; and completely lost. The numbers of CR3 (CD11b/CD18)- and CR4 (CD11c/CD18)-positive cells per glomerular cross-section were counted. At the 1st Bx, no significant difference was found in the number of CR3+ or CR4+ cells between the two groups. At the 2nd Bx, the numbers of both the CR3+ and CR4+ cells were significantly decreased only in group A (P < 0.01). The numbers of CR3+ and CR4+ cells were significantly higher in cases with moderate or marked C3c deposits than in those with no or mild C3c deposits. The intensity of CR1 expression in group B was less than that in group A at both the 1st and 2nd Bx (1st, P < 0.05; 2nd, P < 0.01), and chronological improvement of CR1 expression was observed only in group A. The severity of glomerular injury was increased only in group B (P < 0.01), and was associated with persistent massive proteinuria and hypocomplementaemia. Our results suggest that, in cases with an adverse outcome, a more severe defect of CR1 initially exists and the expression of CR1 is not recoverable chronologically. This irreversible decrease or loss of CR1 may partly contribute to the continuous C3c deposition and intraglomerular infiltration of CR3+ and CR4+ cells.     

Influence of CR3 (CD11b/CD18) expression on phagocytosis of Bordetella pertussis by human neutrophils

CR3 (CD11b/CD18) is expressed on neutrophils, and the engagement of CR3 can promote phagocytosis. CR3 serves as the receptor for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and for the adenylate cyclase toxin (ACT), which blocks neutrophil function. The influence of CR3, FHA, and ACT on the phagocytosis of B. pertussis by human neutrophils was examined. The surface expression and function of CR3 are regulated. Tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) increased CR3 surface expression, but only TNF-alpha increased the ability of neutrophils to phagocytose B. pertussis, suggesting that elevated CR3 expression alone is not sufficient to promote phagocytosis. Purified FHA and pertussis toxin also increased the surface expression of CR3 on neutrophils, while ACT and the B subunit of pertussis toxin did not affect CR3 expression. FHA-mediated attachment to CR3 can lead to phagocytosis, especially in the absence of ACT. FHA mutants failed to attach and were not phagocytosed by neutrophils. Similarly, an antibody to CR3 blocked both attachment and phagocytosis. The addition of exogenous FHA enhanced the attachment and phagocytosis of wild-type B. pertussis and FHA mutants. Mutants lacking the SphB1 protease, which cleaves FHA and allows the release of FHA from the bacterial surface, were phagocytosed more efficiently than wild-type bacteria. ACT mutants were efficiently phagocytosed, but wild-type B. pertussis or ACT mutants plus exogenous ACT resisted phagocytosis. These studies suggest that the activation and surface expression of CR3, FHA expression, and the efficiency of ACT internalization all influence whether B. pertussis will be phagocytosed and ultimately killed by neutrophils.     

Critical role for serum opsonins and complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in phagocytosis of Francisella tularensis by human dendritic cells (DC): uptake of Francisella leads to activation of immature DC and intracellular survival of the bacteria

Francisella tularensis is one of the most infectious human pathogens known. Although much has been learned about the immune response of mice using an attenuated live vaccine strain (LVS) derived from F. tularensis subspecies holarctica (Type B), little is known about the responses of human monocyte-derived immature dendritic cells (DC). Here, we show that optimal phagocytosis of LVS by DC is dependent on serum opsonization. We demonstrate that complement factor C3-derived opsonins and the major complement receptors expressed by DC, the integrins CR3 (CD11b/CD18) and CR4 (CD11c/CD18), play a critical role in this adhesion-mediated phagocytosis. LVS induced proinflammatory cytokine production and up-regulation of costimulatory surface proteins (CD40, CD86, and MHC Class II) on DC but resisted killing. Once taken up, LVS grew intracellularly, resulting in DC death. DC maturation and cytokine production were induced by direct contact/phagocytosis of LVS or interaction with soluble products of the bacteria, and enhanced activation was seen when LVS was pretreated with serum. Sonicated LVS and supernatants from LVS cultures were potent activators of DC, but LVS LPS failed to activate DC maturation or cytokine production. Serum-treated LVS rapidly induced (within 6 h) a number of cytokines including IL-10, a potent suppressor of macrophage functions and down-regulator of Th1-like responses and the Th1 response inducer IL-12. These results suggest that the simultaneous production of an activating (IL-12, IL-1beta, and TNF-alpha) and a suppressing (IL-10) cytokine profile could contribute to the immunopathogenesis of tularemia.     

CR1 (CD35) and CR3 (CD11b/CD18) mediate infection of human monocytes and monocytic cell lines with complement-opsonized HIV independently of CD4.

Peripheral blood and tissue mononuclear phagocytes serve as major viral reservoirs in HIV-infected individuals. We investigated the role of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) in mediating productive infection with complement-opsonized HIV-1 and HIV-2 of cultured normal human peripheral blood monocytes, the promonocytic cell line THP-1, the monocytic cell line Mono Mac 6 and the glial cell line U251-MG. Cells were infected with the HTLV-IIIB strain of HIV-1 or the LAV-2 strain of HIV-2 that had been preopsonized with fresh human normal HIV seronegative serum. Productive infection was assessed by syncytia formation, the MTT cytotoxicity assay and/or release of p24 antigen in culture supernatants. Using suboptimal amounts of virus to infect the cells, we observed a higher and earlier productive infection of the cells with complement-opsonized HIV than with unopsonized virus. The enhancing effect of complement was totally suppressed by blocking CR1 or CR3 function with F(ab)'2 fragments of anti-receptor MoAbs; while blocking of the LFA-1 antigen had no effect. The infection of monocytic cells with complement-opsonized virus occurred independently of CD4 since it was not inhibited by F(ab)'2 fragments of a MoAb against the gp120 binding site of CD4 and since infection also occurred with Mono Mac 6 and U251-MG cells, which lack expression of the CD4 antigen and of CD4 mRNA. These observations suggest that complement may mediate productive infection of cells of the monocytic lineage with 'lymphocytotropic' HIV strains independently of CD4.     

Role of the lectin domain of Mac-1/CR3 (CD11b/CD18) in regulating intercellular adhesion

Leukocytediapedesis requires that Mac-1/CR3-dependent adhesion be regulated so that cells can move from one attachment site to another. The high affinity adhesion state of Mac-1/CR3 is generated when it forms alectin-dependent complex with the receptor for urokinase plasminogen activator (uPAR; CD87). The extensively glycosylated uPAR binds to the same C-terminal lectin domain of CD11b that had previously been shown to prime Mac-1/CR3 for cytotoxic degranulation in response to β-glucan uPAR and β-glucan compete for a lectin site that is near to the CBRM1/23 epitope (residues 943–1047) at the C-terminus of CD11b, and thus the lectin domain is critical to both the adhesion and cytotoxic functions of Mac-1/CR3. Adhesion is reversed when the uPA enzyme is captured by its receptor (uPAR), causing uPAR to bind to CD11b at a second site (residues 424–440) that is in between the N-terminal I-domain and the divalent cation binding region.     

Induction of cell-associated interleukin 1 through stimulation of the adhesion-promoting proteins LFA-1 (CD11a/CD18) and CR3 (CD11b/CD18) of human monocytes.

Serum-free culture of human monocytes in the presence of monoclonal antibodies to the LFA-1 alpha chain (CD11a), CR3 alpha chain (CD11b) or beta chain (CD18) bound to Sepharose induced the dose-dependent production of cell-associated interleukin (IL) 1 activity and of IL 1 alpha and IL 1 beta antigens, but no release of extracellular IL 1 activity or antigen in the culture medium. Triggering of IL 1 production was also observed with insolubilized anti-CD11/CD18 F(ab')2 antibodies. Two cross-linked antibodies recognizing distinct epitopes on the CD11b molecule induced cell-associated IL 1. Soluble antibodies did not induce IL 1 production. The kinetics of induction of IL 1 by stimulation of adhesion-promoting proteins differed from those of IL 1 induction by adhesion to plastic. The lack of induction of IL 1 release by stimulation of the CD11/CD18 molecules resembled the intracellular accumulation of IL 1 induced by lipid A. Induction of IL 1 by adhesive processes may be a mechanism by which T cells trigger IL 1 production by monocytes during antigen presentation.     

Group B streptococcus-induced nitric oxide production in murine macrophages is CR3 (CD11b/CD18) dependent.

Nitric oxide (NO) is produced by murine macrophages in response to cytokines and/or gram-negative bacterial lipopolysaccharide. NO induction by gram-positive bacteria such as group B streptococci (GBS), the major etiologic agents of neonatal pneumonia and meningitis, has received little study. GBS as well as two other gram-positive bacterial species, Staphylococcus aureus and Staphylococcus epidermidis, were found to stimulate NO production in thioglycolate-elicited murine macrophages and in the mouse macrophage cell line J774A.1 in the presence of gamma interferon. Serotype Ia and III GBS were both stimulatory, as were asialo- and type antigen-deficient mutant strains of type III GBS. NO production was dose dependent, inhibitable by L-arginine analogs, and unaffected by polymyxin B. Since phagocytosis by murine and human phagocytes of GBS is dependent on complement receptor type 3 (CR3), the role of CR3 in the NO response to GBS was tested in the CR3-deficient myelomonocytic cell line WEHI-3. GBS did not induce NO, whereas S. aureus or lipopolysaccharide did induce NO in WEHI-3 cells. S. epidermidis, whose nonopsonic phagocytosis is also CR3 dependent, failed to induce NO in WEHI-3 cells. Monoclonal anti-CR3 (anti-CD11b or anti-CD18) in the presence of interferon also induced NO production in thioglycolate-elicited macrophages and in J774A.1 cells but not in WEHI-3 cells. This evidence suggests that ligated CR3 and gamma interferon act synergistically to induce NO production and that CR3 mediates the GBS-induced signal for NO production in interferon-treated macrophages.     

Contribution of CR3, CD11b/CD 18 to cytolysis by human NK cells

The complement receptor CR3 molecule functions in direct intercellular contacts mediated by its beta chain, CD18. Similarly to the Fc receptor (CD16), CR3 is a marker of human natural killer cells. We have shown that opsonization of NK targets with iC3b leads to their increased lytic sensitivity. Opsonization could be achieved by incubating certain B and T cell lines in human serum. The expression of CR2 was a prerequisite for C3 fragment fixation. The CR2 negative cell line, P3HR1 could be opsonized by incubation in human serum when induced to express the EBV envelope glycoprotein gp350. C3b or iC3b could also be deposited artificially on cell surfaces by chemical coupling to surface reactive antibodies. Similarly to the function of macrophages and monocytes, contact with opsonized targets exclusively through the iC3b binding site of CR3 did not seem to trigger NK function. We attempted to clarify the functional role of other CR3 ligands. The beta chain of the molecule, CD18, was essential to the NK effect. The NK targets did not seem to interact with the beta-glucan binding epitope on the alpha chain of CR3, CD11b. On the other hand, the cytolytic function could be enhanced through this epitope with the appropriate ligand.     

Lyn-coupled LacCer-enriched lipid rafts are required for CD11b/CD18-mediated neutrophil phagocytosis of nonopsonized microorganisms

The integrin CD11b/CD18 plays a central role in neutrophil phagocytosis. Although CD11b/CD18 binds a wide range of ligands, including C3bi and beta-glucan, and transmits outside-in signaling, the mechanism of this signaling responsible for phagocytosis remains obscure. Here, we report that lactosylceramide (LacCer)-enriched lipid rafts are required for CD11b/CD18-mediated phagocytosis of nonopsonized zymosans (NOZs) by human neutrophils. Anti-CD11b and anti-LacCer antibodies inhibited the binding of NOZs to neutrophils and the phagocytosis of NOZs. During phagocytosis of NOZ, CD11b and LacCer were accumulated and colocalized in the actin-enriched phagocytic cup regions. Immunoprecipitation experiments suggested that CD11b/CD18 was mobilized into the LacCer-enriched lipid rafts during phagocytosis of NOZs. DMSO-treated, neutrophil-like HL-60 cells (D-HL-60 cells) lacking Lyn-coupled, LacCer-mediated signaling showed little phagocytosis of NOZs. However, loading of D-HL-60 cells with C24 fatty acid chain-containing LacCer (C24-LacCer) reconstructed functional Lyn-associated, LacCer-enriched lipid rafts, and restored D-HL-60 cell NOZ phagocytic activity, which was inhibited by anti-LacCer and anti-CD11b antibodies. Lyn knockdown by small interfering RNA blocked the effect of C24:1-LacCer loading on D-HL-60 cell phagocytosis of NOZs. CD11b/CD18 activation experiments indicated phosphorylation of LacCer-associated Lyn by activation of CD11b. Taken together, these observations suggest that CD11b activation causes translocation of CD11b/CD18 into Lyn-coupled, LacCer-enriched lipid rafts, allowing neutrophils to phagocytose NOZs via CD11b/CD18.     

Nonopsonic phagocytosis of zymosan and Mycobacterium kansasii by CR3 (CD11b/CD18) involves distinct molecular determinants and is or is not coupled with NADPH oxidase activation

Complement receptor type 3 (CR3) was initially described as an opsonic receptor. Subsequently, CR3-mediated lectin-sugar recognition mechanisms have been shown to play a major role in the nonopsonic phagocytosis of several pathogens, among them Mycobacterium tuberculosis. Little is known about the binding and signal transduction mechanisms operating during nonopsonic ingestion through CR3 of different microorganisms. In the present study, we used CHO cells stably transfected with CR3 to show that CR3 was able to mediate internalization of zymosan and pathogenic mycobacteria (Mycobacterium kansasii and Mycobacterium avium) but not that of nonpathogenic species (Mycobacterium smegmatis and Mycobacterium phlei). A combination of mannan and beta-glucan inhibited the phagocytosis of zymosan but had no effect on M. kansasii ingestion. Among six monoclonal antibodies (MAbs) directed against the CD11b subunit of CR3 that decreased zymosan ingestion, only three inhibited M. kansasii phagocytosis. In particular, MAbs known to block the CR3 lectin site affected only internalization of zymosan. Using U937 macrophages, we observed that zymosan ingestion through CR3 induced superoxide production measured by cytochrome c reduction and by translocation of the NADPH oxidase cytosolic component p47phox to the phagosomal membrane, whereas phagocytosis of viable or heat-killed M. kansasii did not. Furthermore, lack of superoxide anion production during phagocytosis of M. kansasii was not due to inhibition of NADPH oxidase per se or superoxide anion scavenging. Together, our results indicate that (i) nonopsonic phagocytosis of zymosan and M. kansasii by CR3 implicates different molecular mechanisms involving multiple and distinct epitopes of CD11b and (ii) CR3 may transduce different cellular responses depending on the sites mediating nonopsonic phagocytosis.     

The function of human NK cells is enhanced by beta-glucan, a ligand of CR3 (CD11b/CD18)

Cells responsible for the natural killer (NK) effect in the human blood can be collected in the low-density lymphocyte subset and the majority of them express CR3. In addition to the iC3b binding site the CR3 molecules possess an epitope which binds beta-glucan. Ligands of this site can deliver activation signals to CR3-carrying monocytes and neutrophils. We found that the function of NK cells was also potentiated by preincubation with beta-glucan. The treatment increased the proportion of target-binding lymphocytes and of the damaged target cells in the conjugates. The monoclonal antibody OKM-1, directed to the beta-glucan-binding site of CR3, abrogated this effect. Another CR3-reactive monoclonal antibody, M522, known to activate monocytes and neutrophils, enhanced the NK function.     

The complement receptor 3, CR3 (CD11b/CD18), on T lymphocytes: activation-dependent up-regulation and regulatory function

The complement receptor 3 (CR3; CD11b/CD18) is present exclusively on leukocytes, particularly on NK cells, monocytes and polymorphonuclear neutrophils. Approximately 10% of peripheral T lymphocytes and, as we found now mainly CD8(+) cells, expressed CD11b. Upon stimulation, however, expression of CD11b was up-regulated also on CD4(+) cells. Stimulation of T cells either by cross-linked anti-CD3 and IL-2 or by mononuclear cells and mitogen yielded up to 28% CD11b(+) T cells. The majority of CD11b(+) T cells also expressed CD56. T cell lines established from healthy donors were also found to express CR3. When restimulated up to 90% of cells became positive for CD11b making those cells an ideal tool for studying the functional role of CD11b. Antibodies to CD11b and bona fide ligands for the complement receptor inhibited the anti-CD3-induced T cell proliferation and as well as IL-2 release. In contrast, proliferation of a CD11b(-) T cell line was not inhibited. Taken together, our data indicate an activation-dependent expression of the complement receptor on T cells and suggest a regulatory function.     

CR3-dependent phagocytosis by murine macrophages: different cytokines regulate ingestion of a defined CR3 ligand and complement-opsonized Cryptococcus neoformans.

Phagocytosis is a fundamental process in innate resistance to infection. We have used the pathogenic yeast Cryptococcus neoformans to study the interaction of this encapsulated organism with murine macrophages in vitro. In the absence of exogenous opsonins the encapsulated yeast is almost totally resistant to ingestion by murine macrophages. Owing to its ability to activate the alternative complement pathway, the anti-phagocytic properties of the polysaccharide capsule can be partially overcome following opsonization in vitro with non-immune mouse serum and subsequent phagocytosis via complement receptors. Here, we demonstrate the importance of the complement receptor type 3 (CR3) in in vitro phagocytosis of the yeast and in in vivo resistance to infection. In vitro, 70% of a population of resident murine macrophages are able to ingest C. neoformans and then only inefficiently (1-2 organisms per cell). Previously we have shown that tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) efficiently enhance ingestion of serum-opsonized encapsulated C. neoformans, and we now show that the cytokines convert a population of resident macrophages to a state where all the cells are competent for ingestion of large numbers of yeasts (6-8 per cell). We also show that these cytokines have a direct effect on CR3, as enhanced levels of complement-opsonized sheep red blood cells (EIgMC) bind to macrophages activated in this way. However, cytokines that have previously been shown to enhance phagocytosis of EIgMC have no effect on ingestion of encapsulated C. neoformans. These results demonstrate that the cytokines regulating CR3-dependent ingestion of C. neoformans are different to those regulating ingestion of EIgMC and reinforce the importance of studying pathogens rather than inert ligands in understanding the regulation of phagocytosis.     

Regulation of CR3 (CD11b/CD18)-dependent natural killer (NK) cell cytotoxicity by tumour target cell MHC class I molecules

Phagocyte and NK cell CR3 functions as both an adhesion molecule and an iC3b receptor mediating cytotoxic responses to microorganisms. Cytotoxic activation of iC3b receptor function requires ligation of both a CD11b I-domain site for iC3b and a lectin site located in the C-terminus of CD11b. Because tumours lack the CR3-binding polysaccharides of bacteria and fungi, iC3b-opsonized tumours do not stimulate CR3-dependent cytotoxicity. Previous studies showed that NK cells could be induced to kill iC3b-opsonized tumours with small soluble β-glucans that bound with high affinity to CR3, bypassing the absence of similar polysaccharides on tumour membranes. Because CR3 signalling requires several tyrosine phosphorylation events, it appeared possible that CR3-dependent killing of autologous tumour cells might be suppressed by NK cell inhibitory receptors for MHC class I (KIR and CD94/NKG2) whose action involves recruitment of SHP-1 and SHP-2 tyrosine phosphatases. In the current study, Epstein–Barr virus (EBV)-transformed B cells were used as targets following opsonization with iC3b. Soluble β-glucan primed CR3 for killing of iC3b-coated B cells, but autologous class I-bearing targets were 84% more resistant than class I-deficient Daudi cells. Blockade of target cell class I with a MoAb specific for a domain recognized by both KIR and CD94/NKG2 resulted in comparable killing of class I⁺ B cells. By contrast, another MoAb to class II had no effect on cytotoxicity. These data suggest that NK cell recognition of class I suppresses CR3/tyrosine kinase-dependent cytotoxicity in the same way as it suppresses cytotoxicity mediated by other tyrosine kinase-linked receptors such as FcγRIIIA (CD16).     

Expression of CD35 (CR1) and CD11b (CR3) on circulating neutrophils and eosinophils from allergic asthmatic children

Complement receptors on neutrophils and eosinophils play a role in activation and adhesion. During asthmatic reactions these receptors have been found elevated on circulating granulocytes. In the present study we compared the expression of CD35 (complement receptor type 1) and CD11b (complement receptor type 3) on neutrophils and eosinophils from asthmatic and non-asthmatic children. This was done in whole blood samples using depolarized light scattering for the discrimination of neutrophils and eosinophils. The non-stimulated expression as well as the upregulated expression of receptors by the chemotactic peptide N-formylmethionyl-leucyl-phenyl-alanine (fMLP) were studied. The results showed that without prior stimulation only the expression of CD35 on neutrophils was significantly elevated in children with asthma (P<0.05). After up-regulation with fMLP, the CD11b expression on neutrophils (P<0.005, fMLP: 0.002 microM) and eosinophils (P<0.05, fMLP: 0.02 microM) was significantly higher in asthmatic children than in the controls. These results indicate that the inducible expression of CD11b on neutrophils and eosinophils from allergic asthmatic children is primed in vivo.     

The roles of complement receptors type 1 (CR1, CD35) and type 3 (CR3, CD11b/CD18) in the regulation of the immune complex-elicited respiratory burst of polymorphonuclear leukocytes in whole blood

The binding of immune complexes (1C) to polymorphonuclear leukocytes (PMN) and the consequent respiratory burst (RB) were investigated in whole blood cell preparations suspended in 75% human serum, using flow cytometry. Blockade of the complement receptor (CR)1 receptor sites for C3b on whole blood cells using the monoclonal antibody (mAb) 3D9 resulted in a 1.9-fold increase in the IC-elicited PMN RB after 5 min of incubation, rising to 3.1-fold after 40 min. This enhancement was not due to increased IC deposition on PMN. Blockade of CR3 abrogated the mAb 3D9-induced rise in RB activity and inhibited the IC binding to PMN in a whole blood cell preparation, with or without mAb 3D9, by approximately 40% from 15–40 min while reducing their RB over 40 min to approximately one third. Blockade of CR1 on either erythrocytes (E) or leukocytes, before mixing the populations, revealed that the potentiation of the RB by mAb 3D9 was associated with abrogation of E-CR1 function, whereas blockade of leukocyte-CR1 had a diminishing effect. Exposure to IC at high concentrations induced release of both specific and azurophilic granule contents from PMN. The latter was CR3 dependent in that blockade of the receptor inhibited the lactoferrin release by one third during 40 min of incubation. In conclusion, CR3 plays a significant role in the IC-mediated generation of an RB and release of specific granules by PMN, while CR1 on whole blood cells, primarily E CR1, restricts the IC-elicited RB in PMN. We propose that CR1 in whole blood promotes the degradation of IC-bound iC3b to C3dg, thereby rendering the IC inaccessible for binding to CR3.     

CR3 (CD11b/CD18) expressed by cytotoxic T cells and natural killer cells is upregulated in a manner similar to neutrophil CR3 following stimulation with various activating agents

CR3 (CD11b/CD18) functions both as an iC3b-receptor and as an adhesion molecule for cellular ligands such as ICAM-1. Although CR3 has been well characterized on phagocytic cells, much less is known about CR3 on lymphocytes. In this study, the expression of CR3 was examined on resting and stimulated B, T, and natural killer (NK) cells by three-color flow cytometry. Biotinylated anti-CR3 mAb and streptavidin-FITC were used in combination with anti-CD3 mAb conjugated with peridinin chlorophyll-a protein (PerCP) and phycoerythrinlabeled mAbs to CD4, CD8, CD19, or CD56. Among resting lymphocytes, CR3 was expressed on nearly all NK cells (CD56⁺CD3^–), 1% of CD4⁺CD3⁺ helper T cells, 7% of CD8⁺CD3⁺ cytotoxic T cells, and 20% of B cells (CD19⁺). Among the 5% of T cells (CD3⁺) expressing CR3, the majority was CD56⁺. Incubation of PBMC for 30 min with PMA induced a three- to fivefold increase in CR3 expression on NK cells and a twofold increase on T cells but did not change the expression of CR3 on B cells. This effect of PMA was not blocked by the presence of cycloheximide, suggesting the presence of cytoplasmic (granule) stores of CR3 in these lymphoid cells resembling those previously reported in neutrophils and monocytes. When PBMC were incubated with rIFN-, rIL-2, -glucan, or high concentrations of LPS, expression of CR3 on NK cells increased significantly, but 4 hr of stimulation was required. Other cytokines (rIFN-, rIL-1, rIL-4, rIL-6, TNF-) and rC5a had no significant effect on CR3 expression. Among NK cells, both the CD56^bright and the CD56^dim cells expressed CR3, and the expression of CR3 on both of these NK cell subsets was increased in a similar manner by PMA. However, rIL-2 stimulated a greater increase in CR3 expression on CD56^bright cells than on CD56^dim cells. These studies suggest that CR3 expressed by NK cells or cytotoxic T cells resembles phagocyte CR3 in that cellular activation stimulates increased surface expression of CR3 derived from cytoplasmic reserves of the receptor.     

A mixed population of immature and mature leucocytes in umbilical cord blood results in a reduced expression and function of CR3 (CD11b/CD18)

Neonatal neutrophils express less membrane and cytoplasmic CR3 (iC3b-receptor, Mac-1, α_Mβ₂-integrin) than do adult neutrophils, and it has been suggested that this renders neonatal neutrophils deficient in diapedesis and bactericidal activity. The reason(s) for this deficiency are unknown. In this study, CR3 expression and the CR3-dependent respiratory burst activity of individual neonatal neutrophils are quantified in comparison with adult leucocytes using flow cytometry. Monocytes and neutrophils are defined as CD14^highCD15^low and CD14^lowCD15^high, respectively. Although neonatal neutrophils bore less CR3 on average than did adult neutrophils, neonatal neutrophils were more heterogeneous and many neonatal neutrophils expressed adult levels of CR3. Because of higher neutrophil concentrations in cord versus adult blood, the calculated number of neutrophils in cord blood expressing high amounts of CR3 was equivalent to that of adult blood. Similar findings were made with monocytes. The size of the CR3-dependent respiratory burst stimulated by particulate β-glucan correlated directly with the expression of CR3 by individual neutrophils. With neonatal and adult neutrophils having comparable CR3 densities, the respiratory burst activities were equivalent. Wright–Giemsa differential staining of the subset of neonatal neutrophils with low CR3 levels isolated by fluorescence-activated cell sorting showed a higher proportion of immature cells than the sorted population expressing high CR3 levels. Therefore, higher proportions of immature cells in cord blood probably explain previous reports of deficient CR3 expression and function. The typical neutrophilia of cord blood may compensate for this apparent deficiency by providing adult concentrations of mature neutrophils.