CR3 (CD11b/CD18) and CR4 (CD11c/CD18) are involved in complement-independent antibody-mediated phagocytosis of Cryptococcus neoformans

IgM and IgA to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) promote complement-independent phagocytosis by macrophages with efficiency comparable to that of IgG1. IgM- and IgA-mediated phagocytosis of C. neoformans was proportional to CR3 expression, inhibited by Abs to CR3 (CD11b/CD18) and CR4 (CD11c/CD18), and dramatically reduced with macrophages of CD18-deficient mice. IgM and IgA promoted ingestion of yeast cells by CHO cells expressing CR3 and CR4. In contrast, IgG1-mediated phagocytosis was only partially inhibited by Abs to CR3 and CR4. Phagocytosis by IgM and IgA but not IgG1 was inhibited by soluble GXM, which binds CD18. Involvement of CR in antibody-mediated complement-independent phagocytosis indicates a new link between innate and adaptive immune systems.     

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.     

Fractalkine/CX3CR1在Ⅳ型狼疮肾炎患者肾组织中的表达

目的： 研究Ⅳ型弥漫增殖型狼疮肾炎、轻微性肾小球病变及正常肾组织中趋化因子fractalkine及其受体CX3CR1的表达，以及与CD68阳性巨噬细胞表达之间的相互关系，探讨fractalkine/CX3CR1在Ⅳ型狼疮肾炎发病中的作用。方法： Ⅳ型狼疮肾炎患者的肾活检组织21例，轻微性肾小球病变患者的肾活检组织18例，光镜下正常的肾组织标本8例。采用免疫组织化学技术检测肾组织中fractalkine、CX3CR1及CD68的表达。结果： （1）正常肾组织和轻微性肾小球病变肾组织中fractalkine基本未见阳性表达；其受体CX3CR1阳性细胞和CD68阳性巨噬细胞仅偶见于肾小球及肾皮质间质中。（2）Ⅳ型狼疮肾炎肾小球和肾皮质间质中CX3CR1阳性细胞和巨噬细胞显著增多，且两者的表达显示高度的相关性（分别为r=0.956，P＜0.01和r=0.965，P＜0.01）。（3）Ⅳ型狼疮肾炎肾组织中fractalkine高表达于皮质肾小管上，fractalkine 阳性皮质肾小管的百分比分别与肾皮质间质CX3CR1阳性细胞数和巨噬细胞数之间高度相关（分别为r=0.720，P＜0.01和r=0.770，P＜0.01）。结论： 提示fractalkine/CX3CR1在Ⅳ型狼疮肾炎的发病机制中可能占有重要地位。     

中国HIV-1暴露未感染者及感染者趋化因子受体CX3CR1的表达研究

目的 通过分析中国HIV-1暴露未感者（exposed semnegative individuals，ESN）及HIV-1感染者外周血中CX3C1^＋CD8^＋／CD3^＋、CX3CR1^＋CD16^＋／CD3^-、CX3CR1^＋CD56^＋／CD3^-细胞百分率及绝对值的变化，探讨CX3CR1受体与HIV-1感染及疾病进展的关系。方法 采集19例ESN、34例未经治疗的HIV-1感染者及18例健康人抗凝静脉血，采用流式细胞仪检测技术，分析计算三色荧光抗体标记的全血中CX3CR1^＋CD8^＋／CD3^＋、CX3CR1^＋CD16^＋／CD3^-、CX3CR1^＋CD56^＋／CD3^-细胞百分率及绝对值。结果 ESNCX3CR1^＋CD8^＋／CD3^＋细胞的百分率是11．05％±6．52％，绝对值是81．16±13．67个／山，显著高于正常对照组（百分率是5．69％±3．94％，绝对值是37．36±8．28个/μl）；HIV-1感染组CX3CR1^＋CD8^＋／CD3^＋细胞的百分率是20．98％±11．88％，绝对值是166．38±138．38个/μl，显著高于正常对照组。ESN的CX3CR1^＋CD16^＋／CD3^-细胞的绝对值是312．49±159．45个／m，显著高于HIV-1感染组（108．83±119．35个／ta）。ESN的CX3CR1^＋CD56^＋／CD3^-细胞的绝对值是316．98±162．56个叫，显著高于HIV-1感染组（100．27±114．57个／ta）。HIV．1感染者的CX3CR1^＋CD16^＋／CD3^-、CX3CR1^＋CD56^＋／CD3^-细胞的绝对值与CIM^＋T淋巴细胞的绝对值呈明显正相关（P〈0．05）。结论CX3CR1^＋CD8^＋／CD3^＋细胞在中国ESN体内起保护作用，而在HIV-1感染者体内发挥有限的保护作用。表达CX3CR1受体的NK细胞可以作为监测HIV-1感染者免疫状况的一个指标。     

The cellular microenvironment regulates CX3CR1 expression on CD8+ T cells and the maintenance of CX3CR1+ CD8+ T cells

Expression levels of the chemokine receptor CX3CR1 serve as high-resolution marker delineating functionally distinct antigen-experienced T-cell states. The factors that influence CX3CR1 expression in T cells are, however, incompletely understood. Here, we show that in vitro priming of naïve CD8⁺ T cells failed to robustly induce CX3CR1, which highlights the shortcomings of in vitro priming settings in recapitulating in vivo T-cell differentiation. Nevertheless, in vivo generated memory CD8⁺ T cells maintained CX3CR1 expression during culture. This allowed us to investigate whether T-cell receptor ligation, cell death, and CX3CL1 binding influence CX3CR1 expression. T-cell receptor stimulation led to downregulation of CX3CR1. Without stimulation, CX3CR1⁺ CD8⁺ T cells had a selective survival disadvantage, which was enhanced by factors released from necrotic but not apoptotic cells. Exposure to CX3CL1 did not rescue their survival and resulted in a dose-dependent loss of CX3CR1 surface expression. At physiological concentrations of CX3CL1, CX3CR1 surface expression was only minimally reduced, which did not hamper the interpretability of T-cell differentiation states delineated by CX3CR1. Our data further support the broad utility of CX3CR1 surface levels as T-cell differentiation marker and identify factors that influence CX3CR1 expression and the maintenance of CX3CR1 expressing CD8⁺ T cells.     

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.     

Altered expression of CD11/CD18 on the peripheral blood phagocytes of patients with tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is characterized by granulomatous lesions made up of epithelioid cells, giant cells and mononuclear leucocytes. Cell-cell adhesion is important in granuloma formation and in the leucocyte migration which accompanies it. We have recently shown increased expression of the adhesion molecules CD11/CD18 (LeuCAMs, beta 2 integrins) on peripheral blood leucocytes from patients with sarcoidosis (Shakoor & Hamblin, 1992). Here we have studied the expression of CD11/CD18 and CD29 (VLA beta 1 integrin) on the peripheral blood leucocytes of 10 TB patients by flow cytometry. The density (expressed as mean fluorescence intensity) of CD11b on monocytes and polymorphs was increased (P < 0.005), as was CD11c (P < 0.005) and CD18 (P < 0.05) on polymorphs. CD11a expression was significantly reduced on polymorphs (P < 0.05). No differences were found in the expression of CD29, the percentages of cells expressing any molecule and, in contrast to sarcoidosis, the density of any molecule on lymphocytes. Although the cytokine tumour necrosis factor (TNF) has been implicated in the process of up-regulation, an ELISA for TNF failed to detect significant levels in plasma. The results suggest increased peripheral phagocyte CD11/CD18 expression is a feature of TB, which may contribute to the pathological processes involved.     

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.     

CR3 (CD11b, CD18): a phagocyte and NK cell membrane receptor with multiple ligand specificities and functions.

The C3 receptor CR3 is expressed on phagocytic cells, minor subsets of B and T cells, and natural killer (NK) cells. It has important functions both as an adhesion molecule and a membrane receptor mediating recognition of diverse ligands such as intercellular adhesion molecule-1 (ICAM-1) and fixed iC3b. The receptor is capable of undergoing an activation event that regulates both its specificity for various ligands and its ability to mediate phagocytosis or extracellular cytotoxicity. Certain bacteria express carbohydrates or lipopolysaccharides (LPS) that can bind to and activate CR3, allowing the receptor to assume its activated state. Soluble beta-glucan derived from the yeast Saccharomyces cerevisiae is a particularly potent stimulator of CR3, and produces an activated state of the receptor that permits neutrophil phagocytosis of iC3b-coated erythrocytes or NK, cell cytotoxicity of iC3b-coated tumour cells, that are normally resistant to NK cells.     

T lymphocyte expression of complement receptor 2 (CR2/CD21): a role in adhesive cell-cell interactions and dysregulation in a patient with systemic lupus erythematosus (SLE).

Complement receptor 2 (CR2, CD21), the receptor for both the C3d,g portion of human complement component C3 and the Epstein-Barr virus, has been recently described on peripheral T cells. By using dual stain flow cytometric analysis, we have also observed that a peripheral T lymphocyte subpopulation of normal healthy donors bears CR2 in a range varying from 1.1 to 23.2% (mean 12.6%) of total CD3+ cells. T lymphocytes from nine patients with inactive SLE expressed CR2 in a similar range. Three patients with active SLE were also studied. One of them, having neuropathy and glomerulonephritis, displayed an expansion of the CR2 T cell subpopulation which reached as much as 89% of total CD3+ cells. To examine potential functional roles of T cell CR2, cells from a Jurkat-derived CR2 expressing T cell line were found to bind in vitro to human CR2-, complement-coated K562 cell targets in a CR2- and complement-dependent fashion. Based on these studies, we hypothesize that CR2 might act to increase adherence of T cells to nucleated target cells bearing C3d,g, a function which may be relevant to cytotoxicity or other T cell activities requiring cell-cell interaction.     

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.     

Cross-linking tumor cells with effector cells via CD55 with a bispecific mAb induces beta-glucan-dependent CR3-dependent cellular cytotoxicity

Complement (C) regulatory proteins decrease the effectiveness of immunotherapeutic anti-cancer antibodies. Bispecific mAb (bi-mAb) that target a tumor antigen and simultaneously inhibit a C regulator increase the effectiveness of such a therapy. Here we investigated the mechanism by which bi-mAb increase tumor cell lysis. Apart from C-dependent cytotoxicity, C activation can lead to complement receptor 3 (CR3)-dependent cellular cytotoxicity (CR3-DCC) by CR3-positive effector cells in the presence of beta-glucan. Here we show that an anti-Ep-CAM*anti-CD55 bi-mAb induced more than threefold higher CR3-DCC (71%) of human colorectal cancer cells compared with anti-Ep-CAM alone (20%). This CR3-DCC was dependent on the binding of the anti-CD55 arm of tumor-bound anti-Ep-CAM*anti-CD55 bi-mAb to effector cell CD55, CR3 priming by beta-glucan and the presence of iC3b on the target cell. Comparable lysis could be obtained in the absence of iC3b, when CR3 and CD55 were cross-linked on the effector cells, suggesting cooperation between CD55 and CR3 in signal transduction. Tumor cells with low antigen expression were effectively lysed via this mechanism in contrast to direct C-dependent cytotoxicity. These data imply that the effectiveness of mAb immunotherapy can be improved using anti-tumor antigen*anti-CD55 bi-mAb and beta-glucan, thereby initiating CR3-DCC as an additional effector mechanism that is efficient for eradication of tumor cells with lower antigen expression.     

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.     

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.     

CD21/CR2的研究进展

补体Ⅱ型受体CD21/CR2是B淋巴细胞膜表面C3d/iC3b受体,在免疫应答中起重要的作用。同时人CD21/CR2也是EB病毒膜表面糖蛋白gp350/gp220、HIV-1等的受体,CD21/CR2以不同的结构域与这些配体结合,表现出多种生物学特性。弄清CD21/CR2的结构和功能,可以指导基础免疫研究,也可明确临床许多疾病的发病机理和治疗方向。