Evidence for the Role of CR1 (CD35), in addition to CR2 (CD21), in Facilitating Infection of Human T Cells with Opsonized HIV

Complement activation by HIV results in the binding of C3 fragments to the gp160 complex and enhanced infection of C3 receptor-bearing target cells. We have studied complement-mediated enhancement of infection of the human CD4-positive T-cell line HPB-ALL which expresses the CR1 (CD35) and CR2 (CD21) receptors for C3. CR1 and CR2 are present on 15% and 40% of normal peripheral blood CD4-positive T lymphocytes respectively. Opsonization of the virus with complement resulted in a 3- to 10-fold enhancement of infection of HPB-ALL cells, as assessed by measuring the release of p24 antigen in culture supernatants throughout the culture period. Blockade of CR2 with cross-linked anti-CR2 monoclonal antibodies decreased infection to the level observed with unopsonized virus. Blocking CR1 reduced complement-mediated infection by 50–80%. Experiments using serum deficient in complement factor I demonstrated that CR1 mediates the interaction between opsonized virus and T cells in addition to its ability to serve as a cofactor for the cleavage of C3b into smaller fragments that interact with CR2. A requirement for CD4 in complement-mediated enhancement of infection was observed with HIV-1 Bru but not with HIV-1 RF. Thus, CR1 and CR2 contribute in an independent and complementary fashion to penetration of opsonized virus into complement receptor-expressing T cells. Involvement of CD4 in infection with opsonized virus depends on the viral strain.     

Mono Mac 1: a new in vitro model system to study HIV-1 infection in human cells of the mononuclear phagocyte series

Throughout the years, most researchers have used continuous cell lines as in vitro models to evaluate the immunopathogenesis of human immunodeficiency virus type-1 (HIV-1) infection. Unfortunately, the most commonly used monocytoid malignant cells have not been shown to adequately mimic primary human monocyte-derived macrophages, at least with respect to HIV-1 infection. The Mono Mac 1 cell line has been defined as a model system for studying biochemical, immunological, and genetic functions of human cells of the monocyte/macrophage lineage. In this study, we have investigated whether Mono Mac 1 represents an in vitro culture system for HIV-1 infection. Flow cytometric analyses revealed that Mono Mac 1 are positive for the HIV-1 primary receptor (CD4), as well as for the coreceptors (CXCR4, CCR5, and CCR3). Infectivity experiments conducted with recombinant luciferase-encoding and fully infectious viruses demonstrated that Mono Mac 1 can support a highly productive infection with both macrophage- and dual-tropic isolates of HIV-1. Furthermore, differentiation of such cells led to a marked increase in virus production. Data from semiquantitative polymerase chain reaction analysis and mobility shift assays indicated that enhanced virus production in differentiated Mono Mac 1 cells was most likely related to an increase in nuclear translocation of NF-kappaB. Mono Mac 1 can thus be considered as a human monocytoid cell line representing a proper in vitro system for studying the complex interactions between HIV-1 and cells of the monocyte/macrophage lineage.     

CR1(CD35) and CR2(CD21) complement C3 receptors are expressed on normal human thymocytes and mediate infection of thymocytes with opsonized human immunodeficiency virus

The present study demonstrates that the C3b receptor CR1 (CD35) and the C3dg/Epstein-Barr virus receptor CR2 (CD21) are expressed by 25% and 70% of normal human thymocytes, respectively. The expression of CR2 extends to both CD1⁺ and CD1^? cells in the thymus. Two subsets of CR2⁺ thymocytes were defined expressing low and high density of the receptor. The CR2⁺⁺ subset represented 20% of CR2⁺ thymocytes and co-expressed the CR1 receptor. CR2⁺⁺ thymocytes expressed an immature CD1^dull, CD3^?, CD4^dull, CD8^?, CD7⁺⁺ phenotype and included a subpopulation of large cells expressing CD34. Twenty percent of thymocytes expressed the CD21 epitope defined by monoclonal antibody BU32, which is involved in the binding of CD23 to CD21. These observations provide a basis for a role for CD21 in the proliferation and differentiation of thymocytes at early stages of maturation. The functionality of CR1 and CR2 on thymocytes was evidenced by the ability of the receptors to mediate infection of cells with complement-opsonized human immunodeficiency virus (HIV). The results may be relevant to the immunopathogenesis of HIV infection.     

CD4-independent binding of HIV-1 to the B lymphocyte receptor CR2 (CD21) in the presence of complement and antibody.

Complement and antibody contribute to infection-enhancement and possible expanded cellular tropism of HIV-1 in vitro through a process requiring complement receptors. Until now, however, the ability of HIV-1 to bind complement receptors has not been documented or characterized. We investigated whether antibody and complement permitted HIV-1 to bind to the B lymphocyte receptor, CR2 (CD21), in an effort to learn more about infection-enhancement, and also because CR2 can mediate B cell proliferation and antigen localization in lymphoid organs in other systems. HIV-1 incubated with antibody and fresh human serum as a source of complement bound approximately 10-fold greater to cells expressing CR2 than to HIV-1-permissive cells lacking this receptor. A similar effect was observed using cells which expressed CR2 but no CD4. This binding was minimal in heat-inactivated and C3-deficient sera, and was significantly reduced by the anti-CR2 MoAb, OKB7, but not by the anti-CD4 MoAb, OKT4a. Thus, complement and antibody acted in concert to facilitate the binding of HIV-1 to CR2 independently of CD4. CD4-independent binding of HIV-1 to CR2 was not sufficient to produce infection in Raji-3 cells. Titres of antibodies mediating CR2 binding correlated with antibody titres as measured by immunofluorescence (P < 0.01) and infection-enhancement (P < 0.05) but were discordant with titres of neutralizing antibodies, a result consistent with the utilization of CR2 for enhanced infection of cells. The ability of complement and antibody to facilitate the binding of HIV-1 to CR2 in the absence of CD4 provides new insights into mechanisms of HIV-1-induced immunopathogenesis and infection-enhancement.     

HIV-1 subverts the complement system in semen to enhance viral transmission

Semen is important in determining HIV-1 susceptibility but it is unclear how it affects virus transmission during sexual contact. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 during sexual contact and have a barrier function as LCs are restrictive to HIV-1. As semen from people living with HIV-1 contains complement-opsonized HIV-1, we investigated the effect of complement on HIV-1 dissemination by human LCs in vitro and ex vivo. Notably, pre-treatment of HIV-1 with semen enhanced LC infection compared to untreated HIV-1 in the ex vivo explant model. Infection of LCs and transmission to target cells by opsonized HIV-1 was efficiently inhibited by blocking complement receptors CR3 and CR4. Complement opsonization of HIV-1 enhanced uptake, fusion, and integration by LCs leading to an increased transmission of HIV-1 to target cells. However, in the absence of both CR3 and CR4, C-type lectin receptor langerin was able to restrict infection of complement-opsonized HIV-1. These data suggest that complement enhances HIV-1 infection of LCs by binding CR3 and CR4, thereby bypassing langerin and changing the restrictive nature of LCs into virus-disseminating cells. Targeting complement factors might be effective in preventing HIV-1 transmission.     

Complement receptor type two (CR2,CR21)

Cellular receptors for complement C3 fragments deposited on antigens are important bricks in the wall defending against microbial pathogens. The part of complement receptor type 2 (CR2; CD21) deals with enhancing humoral immune responses and with long-term trapping of C3d-coated antigen by follicular dendritic cells. CR2 is also pivotal for Epstein-Barr virus (EBV) infection. Here, the current understanding, how CR2 interacts with its ligands C3d, EBV, and CD23 is summarized. The potential to target CR2 for clinical therapy or immunization purposes are discussed.     

Mechanism(s) promoting HIV-1 infection of primary unstimulated T lymphocytes in autologous B cell/T cell co-cultures

Resting CD4(+) T cells in the lymphoid tissue (LT) are essential producers of virions at the beginning of HIV infection in vivo. We previously developed a model that allowed in vitro infection of non-prestimulated T lymphocytes in the presence of autologous B lymphocytes and complement. In this study, we try to clarify the mechanism(s) responsible for virus transmission in unstimulated autologous B cell/T cell co-cultures. Ex vivo analyses of patient plasma samples revealed that HIV was opsonized. Flow cytometry showed that opsonized virus preferentially bound to complement receptor (CR)-2 on B lymphocytes in primary B cell/T cell co-cultures. As indicated by cytokine measurements and transwell experiments, soluble factors seemed to play a minor role in enabling infection. Rather, direct interaction between B and T lymphocytes and direct binding of opsonized virus to CR2 on B cells turned out to be essential for productive infection. Antibodies blocking cell-cell adhesion inhibited p24 antigen production. An anti-CR2 antibody blocking C3d-CR2 binding also significantly reduced viral replication. Since the infection of unstimulated T cells by opsonized primary HIV isolates in the presence of B cells was highly efficient independent of the tropism of the virus, this mechanism may be critical in the pathogenesis of HIV.     

Complement receptor type two (CR2,CR21): a target for influencing the humoral immune response and antigen-trapping

Cellular receptors for complement C3 fragments deposited on antigens are important bricks in the wall defending against microbial pathogens. The part of complement receptor type 2 (CR2; CD21) deals with enhancing humoral immune responses and with long-term trapping of C3d-coated antigen by follicular dendritic cells. CR2 is also pivotal for Epstein-Barr virus (EBV) infection. Here, the current understanding, how CR2 interacts with its ligands C3d, EBV, and CD23 is summarized. The potential to target CR2 for clinical therapy or immunization purposes are discussed.     

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。     

Phenotypic analysis of complement receptor 2+ T lymphocytes: reduced expression on CD4+ cells in HIV-infected persons.

While expression of complement receptor 2 (CR2) (CD21) on some CD4+ cell lines renders them more susceptible to infection by complement-treated human immunodeficiency virus (HIV), coexpression of CR2 and CD4 on peripheral blood lymphocytes has not, until recently, been observed. Several recent studies, however, have found that human T lymphocytes express low levels of CR2. Additionally, complement treatment of HIV before addition to these cells has been reported to increase virus expression in peripheral blood lymphocyte cultures. These findings suggest that complement-mediated enhancement of infection of human T cells could occur in vivo and have prompted us to examine both the phenotypic properties of CD4+CR2+ T cells in healthy persons and the expression of CR2 on CD4+ lymphocytes during HIV infection. As was previously reported, we observed CR2 on a proportion (10-50%) of both CD8+ and CD4+ T cells. Approximately half of CD4+CR2+ cells expressed the memory cell markers CD45RO and CD29, 80% expressed the naive marker CD45RA, while 22% expressed CD25. These values were not substantially different from total CD4+ cells. Stimulation of lymphocytes with phytohaemagglutinin (PHA), OKT3 or calcium ionophore but not with phorbol myristate acetate (PMA) or interleukin-2 (IL-2) decreased expression of CR2 on CD4 cells by half over a 3-day culture period. The per cent of CD4+ cells expressing CR2 was significantly decreased in patients with asymptomatic and symptomatic HIV infection compared to uninfected control donors (P = 0.0001). In contrast, the decrease in CR2 expression was not observed with CD8+ lymphocytes from HIV-infected persons. These results confirm that CR2 is expressed on human T lymphocytes and suggest that a subset of CD4+ lymphocytes is selectively affected in HIV-infected individuals.     

Growth of Chlamydia pneumoniae induces cytokine production and expression of CD14 in a human monocytic cell line.

Chlamydia pneumoniae was able to survive and to multiply in the human monocytic cell line Mono Mac 6. Growth of C. pneumoniae induced production of tumor necrosis factor alpha, interleukin 1beta, and interleukin 6, as well as up-regulation of the CD14 molecule in a time-dependent manner. Infection of monocytic cells and a proinflammatory cytokine response may be important in C. pneumoniae pathogenesis.     

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.     

Infection of human CD4+ rabbit cells with HIV-1: the possibility of the rabbit as a model for HIV-1 infection.

Although human T cell surface glycoprotein CD4 is the cellular receptor for human immunodeficiency virus 1 (HIV-1), the introduction of the human CD4 gene into murine cells does not render them susceptible to HIV-1 infection. Here we have established rabbit transfectant cell lines expressing human CD4 on the cell surface and demonstrated that the CD4+ rabbit transfectants could be readily infected by HIV-1 by co-cultivating with a HIV-1-infected human MOLT-4 T cell line (MOLT-4/HIV). Avid syncytia formation was observed upon co-cultivation and the syncytia abundantly produced HIV-1 mature particles, as revealed by electron microscopy. A significant increase of HIV-1 p24 antigen was also detected in the culture supernatant. The syncytia formation was blocked by pretreating the transfectant with anti-human CD4 or by pretreating the MOLT-4/HIV with anti-HIV-1 serum obtained from an infected individual, indicating that the syncytia formed as a result of the interaction of human CD4 on the rabbit transfectant with the HIV-1 envelope protein expressed on MOLT-4/HIV. In contrast, only a very small proportion of the rabbit transfectants expressed HIV-1-specific antigens upon infection with an HIV-1 stock. This may indicate that, although rabbit cells have partially acquired susceptibility to HIV-1 by transfection of human CD4 gene, rabbit cells may further require such a molecule as might be provided by MOLT-4 to become fully susceptible to HIV-1 infection. The possibility of the rabbit as a model for HIV-1 infection is also discussed.     

Virus-cell membrane fusion does not predict efficient infection of alveolar macrophages by human immunodeficiency virus type 1 (HIV-1).

Alveolar macrophages (AM) are the principal target cells for HIV-1 in lung tissue. To investigate the mechanisms of HIV-1 infection and efficient replication in these cells we isolated AM from 14 HIV-1 negative donors and exposed them to two virus isolates, either N1T, which replicates well in T lymphocytic and monocytic cell lines, or ADA, a monocytotropic virus. Membrane fluorescence dequenching assays demonstrated that HIV-1/N1T fuses efficiently with AM plasma membranes at neutral pH and that this interaction requires cellular CD4. Despite efficient fusion, AM from eight of 14 donors were not susceptible to productive infection with N1T. In contrast, ADA replicated in all AM populations tested. Soluble CD4 blocked infection of AM by either N1T or ADA, indicating that, like membrane fusion, entry of infectious virus requires an interaction with cellular CD4. Analysis of HIV-1 DNA accumulation in infected cells by enzymatic amplification revealed that productive infection by ADA correlated with a high HIV-1 DNA copy number and abortive infection by N1T was characterized by little or no stable cDNA. These studies suggest that the differences between the two HIV-1 strains studied in their ability to replicate in AM reside in phases of the virus life cycle that follow virus-cell fusion.     

Purified recombinant CD4 inhibits HIV-1 infection of peripheral blood macrophages.

Purified recombinant soluble CD4 (rCD 4) is a new antiviral agent which has been shown to block HIV infection of lymphocytic and monocytic cell lines as well as peripheral blood mononuclear cells. The authors investigated here the ability of rCD4 to inhibit HIV-1 infection of peripheral blood macrophages. In this system, purified preparations of rCD4 provided, at concentrations above 10 micrograms/ml, a complete protection against a challenge with the monocytotropic strain HIV-1 Ba-L. Also, anti-CD 4 monoclonal antibodies directed at the V1 domain of the CD 4 molecule blocked infection of macrophages by HIV. Recombinant CD 4 is therefore a potent antiviral agent able to prevent HIV-1 infection of peripheral blood macrophages, and the CD 4 molecule is likely to be the main HIV receptor on these cells.     

Human cytomegalovirus downregulates complement receptors (CR3, CR4) and decreases phagocytosis by macrophages

Human cytomegalovirus (HCMV) infection is associated with an increased susceptibility to opportunistic infections. Although the subversion of adaptive immune responses has been extensively studied, the consequences of HCMV infection on natural immune responses are not well documented. A striking selective downmodulation of CD11b/CD18 (CR3) or CD11c/CD18 (CR4) was found upon HCMV infection, on two models, the monocytic THP-1 cell line and monocyte- derived macrophages. HCMV-infected macrophages have an altered adhesion/phagocytic capacity to Candida albicans, a pathogen responsible for some opportunistic infections in immunocompromised patients. These results suggest a new mechanism implicated in the augmentation of opportunistic infections in HCMV patients.     

Antigen stimulation induces HIV envelope gp120-specific CD4(+) T cells to secrete CCR5 ligands and suppress HIV infection

CD4(+) T cells are critical for effective immune responses against HIV, but they are also the main cell type targeted by the virus. To investigate the key factors that could protect these cells from infection, we evaluated the capacity of HIV gp120-specific human CD4(+) T cells to produce chemokines that inhibit HIV and determined their contribution in suppressing infection in the cells. Antigen stimulation of the CD4(+) T cells elicited production of high amounts of CCR5 chemokines MIP-1alpha (CCL3), MIP-1beta (CCL4), and RANTES (CCL5). Production of these CCR5 ligands was more readily and reproducibly detected than that of IFN-gamma or IL-2. Importantly, in association with secretion of the CCR5 ligands, antigen stimulation made these CD4(+) T cells more resistant to CCR5-tropic HIV-1. Conversely, in the absence of antigen stimulation, the cells were readily infected by the virus, and after infection, their capacity to produce MIP-1beta and IFN-gamma rapidly declined. Thus, vaccines that trigger HIV-specific CD4(+) T cells to elicit robust and rapid production of anti-viral chemokines would be advantageous. Such responses would protect virus-specific CD4(+) T cells from HIV infection and preserve their critical functions in mounting and maintaining long-lasting immunity against the virus.     

Downregulation of CCR5 on activated CD4 T cells in HIV-infected Indians

BACKGROUND: HIV infection in India is unique as it occurs predominantly by CCR5-utilizing isolates that exhibit no co-receptor switch. OBJECTIVES: To study HIV-1 co-receptor dynamics on T cells and monocytes following viral infection. STUDY DESIGN: HIV co-receptor expression was evaluated by flow cytometry on various cell subsets in HIV-infected Indians and in vitro in human peripheral blood mononuclear cells infected with CCR5- or CXCR4-utilizing HIV-1. Transfection of the T cell line CEM-CCR5 (which expresses CD4, CCR5 and CXCR4) with HIV-1 Nef or Vpu expression vectors, or treatment with recombinant soluble gp120 from CCR5- and CXCR4-tropic HIV-1, was carried out to determine their effects on co-receptor expression. RESULTS: Indian HIV patients had fewer CD4(+)CCR5(+) T cells and CCR5-expressing activated CD4(+) T cells, but higher CXCR4-expressing activated CD4(+) T cells compared with controls. Expression of CCR5 was not different on monocytes in HIV patients as compared to controls. The CCR5 downregulation on T cells was HIV infection specific and was governed by the co-receptor-utilization phenotype of the virus. The Nef and soluble gp120 proteins induced CCR5 downregulation, the latter in a co-receptor-utilization phenotype specific manner. CONCLUSIONS: The HIV-1 co-receptor dynamics in Indian patients is distinct from western patients and depends upon the virus surface protein. We propose this to be a viral survival strategy.