Cell Surface Expression Of CCR5 Research Articles

Schistosoma mansoni soluble egg antigen (SEA) and recombinant Omega-1 modulate induced CD4+ T-lymphocyte responses and HIV-1 infection in vitro

Parasitic helminths evade, skew and dampen human immune responses through numerous mechanisms. Such effects will likely have consequences for HIV-1 transmission and disease progression. Here we analyzed the effects that soluble egg antigen (SEA) from Schistosoma mansoni had on modulating HIV-1 infection and cytokine/chemokine production in vitro. We determined that SEA, specifically through kappa-5, can potently bind to DC-SIGN and thereby blocks DC-SIGN mediated HIV-1 trans-infection (p<0.05) whilst not interfering with cis-infection. DCs exposed to SEA whilst maturing under Th2 promoting conditions, will upon co-culture with naïve T-cells induce a T-cell population that was less susceptible to HIV-1 R5 infection (p<0.05) compared to DCs unexposed to SEA, whereas HIV-1 X4 virus infection was unaffected. This was not observed for DCs exposed to SEA while maturing under Th1 or Th1/Th2 (Tmix) promoting conditions. All T-cell populations induced by SEA exposed DCs demonstrate a reduced capacity to produce IFN-γ and MIP-1β. The infection profile of T-cells infected with HIV-1 R5 was not associated with down-modulation of CCR5 cell surface expression. We further show that DCs maturing under Tmix conditions exposed to plant recombinant omega-1 protein (rω-1), which demonstrates similar functions to natural ω-1, induced T-cell populations that were less sensitive for HIV-1 R5 infection (p<0.05), but not for X4 virus infection. This inhibition associated again with a reduction in IFN-γ and MIP-1β expression, but additionally correlated with reduced CCR5 expression. We have shown that SEA parasite antigens and more specifically rω-1 can modulate HIV-1 infectivity with the potential to influence disease course in co-infected individuals.

Transcriptional down-regulation of ccr5 in a subset of HIV+ controllers and their family members.

HIV +Elite and Viremic controllers (EC/VCs) are able to control virus infection, perhaps because of host genetic determinants. We identified 16% (21 of 131) EC/VCs with CD4 +T cells with resistance specific to R5-tropic HIV, reversed after introduction of ccr5. R5 resistance was not observed in macrophages and depended upon the method of T cell activation. CD4 +T cells of these EC/VCs had lower ccr2 and ccr5 RNA levels, reduced CCR2 and CCR5 cell-surface expression, and decreased levels of secreted chemokines. T cells had no changes in chemokine receptor mRNA half-life but instead had lower levels of active transcription of ccr2 and ccr5, despite having more accessible chromatin by ATAC-seq. Other nearby genes were also down-regulated, over a region of ~500 kb on chromosome 3p21. This same R5 resistance phenotype was observed in family members of an index VC, also associated with ccr2/ccr5 down-regulation, suggesting that the phenotype is heritable.

CCR5 promoter activity correlates with HIV disease progression by regulating CCR5 cell surface expression and CD4 T cell apoptosis

CCR5 is the major co-receptor for HIV and polymorphisms in the CCR5 gene as well as promoter region that alter cell surface expression have been associated with disease progression. We determined the relationship between CCR5 promoter polymorphisms and CD4 decline and other immunopathological features like immune activation and CD4+ T cell apoptosis in HIV patients. CCR5 promoter haplotype HHC was significantly associated with higher CD4 counts in patients. The relative promoter activity (RPA) of each haplotype was determined in vitro and combined promoter activity based on both alleles (CRPA) was assigned to each patients. Interestingly, CCR5 CRPA correlated inversely with CD4 counts and CD4:CD8 ratio specifically in viremic patients. In normal individuals, the CRPA correlated with the number of CCR5+ CD4+ T cells in the peripheral blood suggesting an effect on CCR5 expression. In a subset of high viremic patients harboring R5 tropic HIV, there was a strong correlation between CCR5 CRPA and both CD4 counts and CD4 T cell apoptosis. Our study demonstrates that, CCR5 promoter polymorphisms correlate with CD4 T cell loss possibly by regulating CD4 T cell apoptosis in HIV patients. Furthermore, assigning CRPAs to each patient is a new method of translating genotype to phenotype.

Marked differences in CCR5 expression and activation levels in two South African populations

The chemokine receptor CCR5 is pivotal in determining an individual's susceptibility to HIV-1 infection and rate of disease progression. To establish whether population-based differences exist in cell surface expression of CCR5 we evaluated the extent of CCR5 expression across all peripheral blood cell types in individuals from two populations, South African Africans (SAA) and South African Caucasians (SAC). Significant differences in CCR5 expression, both in number of CCR5 molecules per cell (density) and the percentage of CCR5-expressing cells, were observed between the two study groups, within all cell subsets. Most notably, the percentage of all CCR5(+) cell subsets was significantly lower in SAC compared with SAA individuals (P < 0·01) among natural killer (NK) -cell subsets (CD56(+) , CD16(+) CD56(+) and CD56(dim) ) whereas CCR5 density was significantly higher in SAC compared with SAA individuals in CCR5(+) CD8(+) T-cell subsets and CCR5(+) NK-cell subsets (CD56(+) , CD16(+) CD56(+) and CD56(dim) ) (all P < 0·05). These relationships were maintained after exclusion of CCR5Δ32 heterozygous individuals (n = 7) from the SAC dataset. The SAA individuals exhibited significantly higher cell activation levels, as measured by HLA-DR expression, than SAC individuals in CD4(+) T-cell subsets (P = 0·002) and CD56(+) NK-cell subsets (P < 0·001). This study serves to demonstrate that ethnically divergent populations show marked differences in both cell activation and CCR5 expression, which are likely to impact on both susceptibility to HIV-1 infection and the rate of HIV-1 disease progression.

CCR5 as Target for HIV-1 Gene Therapy

Acquired immune deficiency syndrome (AIDS) is caused by a lentivirus, human immunodeficiency virus type-1 (HIV-1). Viral entry is mediated by specific interaction of the viral envelope (Env) glycoprotein with a cell surface molecule CD4 which serves as the primary receptor and a chemokine (C-C or C-X-C motif) receptor CCR5 or CXCR4 which serves as a co-receptor. The viral Env, the cellular CD4 receptor, or the CCR5/CXCR4 co-receptors may be the targets of therapeutic interventions. Compared to the high variability of the viral Env protein, lack of variability in the CD4 receptor and the CCR5 or CXCR4 co-receptor makes them better targets to prevent viral entry. Downregulation of CD4 or CXCR4 is likely to have harmful consequences for the immune function or cellular maturation and homing. In contrast, individuals who lack functional CCR5 have no apparent immune defects, and show decreased susceptibility to HIV-1 infection and delayed progression to AIDS. CCR5 is essential for HIV-1 infection through all routes of transmission. Therefore, its downregulation may not only prevent disease progression, but also the spread of HIV-1 transmission. To block CCR5 function, a number of molecules were developed, including low molecular weight compounds, chemokines, N-terminally-modified chemokine analogues, chemokine-derived molecules, chemokine-based synthetic peptides, and anti-CCR5 monoclonal antibodies. Gene therapy strategies were developed using intrakines and intrabodies to prevent cell surface expression of CCR5 and zinc finger-nucleases, or using small interfering RNAs, antisense RNAs, or ribozymes to decrease co-receptor synthesis. This review describes the importance of targeting CCR5 and summarizes the status of various anti-CCR5 gene therapy strategies.

Inhibition par la sérotonine de l'infection par VIH-1 des macrophages en culture primaire: rôle du sous-type 5-HT1A des récepteurs sérotoninergiques

The neurotransmitter 5-hydroxytryptamine (5-HT), commonly known as serotonin, is released at peripheral sites from activated platelets. At inflammatory sites, macrophages and lymphocytes could be exposed to 5-HT concentrations up to 100 microM. Moreover, 5-HT could modulate cytokine secretion by monocytes/macrophages and immune functions through the uptake of 5-HT at these inflammatory sites from T cells and dendritic cells. HIV infection is also under the control of inflammatory processes (including T cell proliferation and cytokines secretion). On this basis, we studied explored herein the effects of 5-HT on HIV-1/Ba-L (macrophage-tropic virus) replication in primary cultures of human macrophages. This pharmacological study with isotype-selective receptor agonists and antagonist allowed us to show that the 100 microM 5-HT concentration via 5-HT(1A) subtype receptors could decrease HIV replication. This observation was associated with an increase of MIP-1alpha secretion such as an increase of MIP-1alpha mRNA production and with a decrease of HIV-coreceptor CCR5 cell surface expression. Our results point out for the first time the inhibitory effects of 5-HT on HIV replication in primary culture of human macrophages via activation of 5-HT(1A) subtype receptors.

Chemokine receptor CCR5 in interferon-treated multiple sclerosis

To study the relationship between CC chemokine receptor CCR5 expression and disease activity in multiple sclerosis (MS) patients treated with beta-interferon (IFN-beta). The CCR5 Delta32 allele and a CCR5 promoter polymorphism associated with cell surface expression of CCR5 were analyzed in 109 patients with relapsing-remitting MS treated with IFN-beta who were followed clinically for 1 year. Cellular CCR5 expression was measured by flow cytometry. Patients with MS had a higher percentage of CCR5-positive monocytes than healthy controls. Increased monocyte expression of CCR5 correlated weakly with an increased short-term relapse risk but there was no relationship between CCR5 Delta32 allele and CCR5 promoter polymorphism genotypes and relapse risk. The results do not support a major role of CCR5 in the pathogenesis of relapses in MS patients treated with IFN-beta, but it is possible that monocyte CCR5 expression may be used as a marker of disease activity.

Production of Specific mRNA Transcripts, Usage of an Alternate Promoter, and Octamer-Binding Transcription Factors Influence the Surface Expression Levels of the HIV Coreceptor CCR5 on Primary T Cells

Surface levels of CCR5 on memory CD4(+) T cells influence HIV-1/AIDS susceptibility. Alternative promoter usage results in the generation of CCR5 mRNA isoforms that differ based on whether they contain or lack the untranslated exon 1. The impact of exon 1-containing transcripts on CCR5 surface expression is unknown. In this study, we show that the increased cell surface expression of CCR5 on primary T cells is associated with selective enrichment of exon 1-containing transcripts. The promoter that drives exon 1-containing transcripts is highly active in primary human T cells but not in transformed T cell lines. The transcription factors Oct-1 and -2 inhibit and enhance, respectively, the expression of exon 1-containing transcripts and CCR5 surface levels. However, polymorphisms at homologous octamer-binding sites in the CCR5 promoter of nonhuman primates abrogate the binding of these transcription factors. These results identify exon 1-containing transcripts, and the cis-trans factors that regulate the expression levels of these mRNA isoforms as key parameters that affect CCR5 surface expression levels, and by extension, susceptibility to HIV/AIDS among humans, and possibly, the observed interspecies differences in susceptibility to lentiviral infection.

Cell surface expression of CCR5 and other host factors influence the inhibition of HIV-1 infection of human lymphocytes by CCR5 ligands

Several CCR5 ligands, including small molecules and monoclonal antibodies (MAbs), are being developed as therapies for infection with strains of human immunodeficiency virus type 1 (HIV-1) that use CCR5 for entry (R5 viruses). The efficacy of such therapies could be influenced by inter-individual differences in host factors, such as CCR5 expression levels. To study this, we used peripheral blood mononuclear cells (PBMCs) from humans and rhesus macaques. The half-maximal inhibitory concentrations (IC 50) of the small-molecule CCR5 ligands CMPD167, UK427,857 and SCH-D, and of the PRO 140 MAb, differ by > 2 logs in a donor-dependent manner. We studied this variation by using flow cytometry to measure CCR5 expression on PBMCs from six of the human donors: the IC 50 values of both SCH-D and PRO 140 correlated with CCR5 expression ( R 2 = 0.64 and 0.99, respectively). We also determined the efficacy of the CCR5 ligands against HIV-1 infection of HeLa-derived cell lines that express CD4 at the same level but vary 2-fold in CCR5 expression (JC.48 and JC.53 cells). The moderately greater CCR5 expression on the JC.53 than the JC.48 cells was associated with proportionately higher median IC 50 values for all four CCR5 ligands but not for a soluble CD4-based inhibitor or a non-nucleoside reverse transcriptase inhibitor. We conclude that differences in CCR5 expression on human PBMCs, which can be affected by CCL3L1 gene dose, may influence the antiviral potency of CCR5 ligands in vitro, but other host factors are also likely to be involved. These host factors may affect the clinical activity of CCR5 inhibitors, including their use as topical microbicides to prevent HIV-1 transmission.

Aging sensitizes mice to behavioral deficits induced by central HIV-1 gp120

The number of older adults with HIV-1 disease is increasing but little is known about how age influences behavioral deficits associated with HIV-1 infection. The purpose of this study was to determine in a murine model if aging influenced sickness behavior following central injection of HIV-1 gp120. In initial studies, behavioral deficits induced by acute and repeated intracerebroventricular (ICV) injection of gp120 were greater in aged mice than in adults. Furthermore, repeated ICV injection of gp120 increased hippocampal levels of IL-1β and IL-6 mRNA in aged mice but not in adults. To determine if IL-6, which is elevated in aged brain, affects expression of the gp120-binding target, CCR5, microglia (BV-2 cell line) were incubated with increasing concentrations of IL-6. Cell surface expression of CCR5 was increased by IL-6 in a dose-dependent manner. Additionally, IL-6 increased gp120-dependent chemotaxis. These results suggest that aging increases the sensitivity of mice to behavioral deficits caused by ICV gp120, perhaps by increasing expression of CCR5 and augmenting production of cytokines.

Interaction between the CCR5 chemokine receptors and microbial HSP70

Evidence is presented that the microbial 70-kD heat shock protein (HSP70) binds to CCR5 chemokine receptors in CCR5-transfected cell lines and in primary human cells. Significant CCR5-mediated calcium mobilization was stimulated by HSP70 and inhibited with TAK 779, which is a specific CCR5 antagonist. HSP70-mediated activation of the p38 MAPK phosphorylation signaling pathway was also demonstrated in CCR5-transfected HEK 293 cells. Direct binding of three extracellular peptides of CCR5 to HSP70 was demonstrated by surface plasmon resonance. Functional evidence of an interaction between HSP70, CCR5 and CD40 was shown by enhanced production of CCL5 by HEK 293 cells transfected with both CD40 and CCR5. Primary monocyte-derived immature DC stimulated with HSP70 produced IL-12 p40, which showed dose-dependent inhibition of >90% on treatment with both TAK 779 and anti-CD40 mAb. Stimulation of IL-12 p40 or TNF-alpha by HSP70 was related to the differential cell surface expression of CCR5 in primary human immature and mature DC, and those with the homozygous triangle DeltaDelta32 CCR5 mutation. These findings may be of significance in the interaction between HSP70 and immune responses of CCR5+ T cells in HIV-1 infection, as well as in inflammatory bowel disease.

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

CCR5 is the chemokine co-receptor for R5-tropic human immunodeficiency virus type 1 (HIV-1) isolates most often associated with primary infection. We have developed an HIV-1 self-inactivating vector, CAD-R5, containing a CCR5 single-chain antibody (intrabody) gene, which when expressed in T-cell lines and primary CD4+ T cells disrupts CCR5 cell surface expression and provides protection from R5-tropic isolate exposure. Furthermore, CAD-R5 intrabody expression in primary CD4+ T cells supports significant growth and enrichment over time during HIV-1-pulsed dendritic cell-T-cell interactions. These results indicate that CCR5 intrabody-expressing CD4+ T cells are refractory against this highly efficient primary route of infection. CD34+ cells transduced with the CAD-R5 vector gave rise to CD4+ and CD8+ thymocytes in non-obese diabetic (NOD)/ severely combined-immunodeficient (SCID)-human thymus/liver (hu thy/liv) mice, suggesting that CCR5 intrabody expression can be maintained throughout differentiation without obvious cellular effects. CD4+ T cells isolated from NOD/SCID-hu thy/liv mice were resistant to R5-tropic HIV-1 challenge demonstrating the maintenance of protection. Our findings demonstrate delivery of anti-HIV-1 activity through CCR5 intrabodies in primary CD4+ T cells and CD34+ cell-derived T-cell progeny. Thus, gene delivery strategies that provide a selective survival and growth advantage for T effector cells may provide a therapeutic benefit for HIV-1-infected individuals who have failed conventional therapies.

Chemokine Receptor CCR5 Expression in Conjunctival Epithelium of Patients With Dry Eye Syndrome

To characterize chemokine receptor CCR5 expression on the conjunctival epithelium in dry eye syndromes. Conjunctival impression cytology samples were obtained from normal subjects (n = 15) and patients with dry eye syndrome (n = 45). Cells were harvested from impression cytology samples, and flow cytometry was performed to quantitatively analyze the cell surface expression of chemokine receptor CCR5. Characterization of CCR5-positive cells was done by 2-color flow cytometry using fluorescein-conjugated anti-CCR5 and phycoerythrin-conjugated anti-CD45 antibodies (where CD45 is a marker for bone marrow-derived cells). To study CCR5 messenger RNA transcripts, real-time polymerase chain reaction was done on RNA isolated from the impression cytology samples of normal subjects (n = 5) and patients with dry eye syndrome (n = 14). We found significant up-regulation in cell surface expression of CCR5 in patients with both aqueous tear-deficient and evaporative forms of dry eye syndrome (P<.001). The real-time polymerase chain reaction results (for messenger RNA) corroborated the flow cytometry data (for protein). The majority of the cells expressing CCR5 were non-bone marrow-derived resident epithelial cells of the conjunctiva. Our findings suggest that CCR5 up-regulation is significantly associated with dry eye syndrome-associated ocular surface disease. Clinical Relevance Chemokine receptor CCR5 or its ligands may serve as useful targets for modulation of tissue immunoinflammatory responses in dry eye syndromes.

973. Targeting of CCR5 through Intrabody Mediated HIV-1 Vector Gene Delivery Is Sufficient for Blocking HIV-1-Infection and Confers a T-Cell Survival and Growth Advantage

Top of pageAbstract With the emergence of HAART resistant HIV-1s, there is a need for additional therapies to preserve T-effector cells and reduce viral burden. Moreover, since it may be impossible to protect all susceptible cells in an individual through gene delivery, it is therefore necessary to develop and verify that genetic means of protection against HIV-1 provides a cellular survival and growth advantage. To this purpose we have developed single chain antibodies to CCR5 which targets protein. Efficacy of HIV-1 protection and enrichment was assessed by free viral and HIV-1 loaded dendritic cell challenge. HIV-1 vector delivery of CCR5-intrabody (termed the CAD-R5 vector), coupled to a KDEL endoplasmic retention sequence, to primary CD4 T-cells efficiently disrupted CCR5 cell surface expression 4-fold as determined by mean fluorescence intensity (MFI) as compared to a control vector without the intrabody, 8 and 34 MFI, respectfully. CAD-R5 transduced primary CD4 T-cells were resistant to robust, free, R5-tropic SF162 challenges as shown by 50 to 60-fold reductions in HIV-1 p24 concentrations. Moreover, CCR5-intrabody gene expressing CD4 T-cells, as compared to cells with a control vector, demonstrated a selective advantage and enriched from 2-10-fold in dendritic cell cultures. Similar levels of HIV-1 protection were seen in CD34 cell derived macrophages; 3-weeks after challenge with SF162, macrophages expressing CCR5-intrabody generated 42-, 57-, and 52-fold less p24 as compared to parental and control vector containing macrophages. Thus, CCR5 intrabody expression drastically decreased viral loads in CD4 T-cells and macrophages. To assess hematopoietic developmental alterations from CCR5-intrabody expression, CD34 cells were transduced and colony forming (CFU) and macrophage assays were performed. No differences were seen in numbers/types of macrophages or CFUs produced when comparing parental, control vector, and intrabody expressing cells (n=3). To assess in vivo correlates of hematopoietic function and intrabody-mediated protection, CAD-R5 and control vector transduced, fetal liver derived CD34 cells were engrafted in human thymi implants in SCID-hu thy/liv mice (n = 2, 2 groups), 6-weeks later the thymi displayed an average of 25% reporter gene expressing T-cells, similar to controls. Development was not altered as compared to controls as determined by CD4/CD8 profile. Furthermore, CCR5-intrabody expressing thymocytes were also highly resistant to ex vivo R5-tropic HIV-1 challenge. These results validate the efficacy of HIV-1 delivered CCR5-intrabody mediated protection from HIV-1 challenges in primary T-cells and macrophages and, importantly, demonstrates a survival and growth advantage of protected CD4 T-cells. The findings underscore the potential advantage of intrabody gene delivery to CD34 cells, which allows differentiation of protected T-cells, macrophages, and dendritic cells.

Lentiviral CCR5 Intrabody Gene Delivery Provides Protection and Enrichment during CCR5-Tropic Infection.

The molecular mechanism of human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep mechanism. The viral envelope glycoproteins (env) binds first to CD4 and subsequently interacts with the V3 loop with a chemokine receptor, CCR5 or CXCR4, triggering the fusion event. Several findings suggest that viruses using CCR5 for entry (R5-tropic HIV-1) is the predominant species transmitted among patients. Importantly, CCR5 expression levels determine disease progression. CCR5 does not seem to be necessary for normal cell function, since individuals with a homozygous mutation (Δ32) do not appear to have clinical, immune alterations. Furthermore, these individuals are highly protected against transmission of R5-tropic HIV-1. Therefore, intervention strategies aimed at altering or blocking CCR5 expression may be beneficial for cellular protection and provide a clinical benefit against HIV-1 infection. We have constructed an HIV-1 derived vector, CAD-R5, expressing an intracellular single-chain antibody (intrabody) specific for CCR5 coupled to a KDEL endoplasmic reticulum retention signal. Intrabody expressing primary T-cells efficiently disrupted CCR5 cell surface expression with a 4.3-fold reduction in CCR5 mean fluorescence intensity (MFI) as compared to the control vector without the intrabody gene (7.9 and 33.6 MFI, respectfully). CAD-R5 transduced primary CD4 T-cells expressing the intrabody gene were resistant to R5-tropic HIV infection as shown by a 50 to 60-fold reduction in HIV-1 p24 concentration. Moreover, intrabody gene expressing cells demonstrated a selective advantage and enriched by 6.4-fold in a population of infected cells as compared to uninfected or infected cells containing vector without the intrabody gene. The SCID-human mouse model, produced by conjoining fetal human thymus and liver under the renal capsule, has become a staple for in vivo testing of T cell anti-retroviral therapy. When CAD-R5 transduced, fetal liver derived CD34+ stem cells were used for reconstitution of the human implants in SCID-hu mice (n = 2, 2 groups), 6-weeks later the human thymi displayed an average of 25% reporter gene expressing T cells. Thymocyte development was not altered by vector integration or loss of CCR5 cell surface expression as determined by the CD4+/CD8+ staining profile. Importantly, CCR5 intrabody expressing thymocytes were also highly resistant to ex vivo R5-tropic HIV-1 challenge with a 3-fold reduction in viral load. These results validate the efficacy of lentiviral delivered CCR5 intrabody mediated protection from R5-tropic HIV-1. The findings also underscore the potential advantage of intrabody gene delivery to CD34+ stem cells, which allows differentiation of protected T cell progeny.

CD34+ Cell Derived Macrophages Are Protected from HIV-1 Challenge by Intrabody-Mediated Reduction of CCR5.

The three main coreceptors that HIV-1 uses for viral entry are CD4 and the chemokine receptors CXCR4 and/or CCR5. Viral tropism dictates which chemokine receptors are used for viral entry. Naturally occurring CCR5 gene polymorphisms resulting in gene deletion (delta 32) results in loss of receptor expression and provides protection from CCR5 using viruses. There are no known deleterious effects of CCR5 loss in mice or in humans. The focus of our studies is to limit infection of myeloid cells by CCR5 using HIV-1s by decreasing CCR5 cell surface expression. We have successfully accomplished this by using HIV-1 vectors to deliver an intrabody directed against CCR5. The CCR5-specific intrabody gene, containing the endoplasmic KDEL sequence, produces a single chain antibody that sequesters CCR5 in the endoplasmic reticulum. We show that >= 93% of THP-1s, a myelomonocytic cell line, transduced with CCR5 intrabody genes stably expressed intrabodies for 6 months. Of cells expressing intrabody, CCR5 was decreased at least 80% on the cell surface. In macrophages derived from CD34+ cells, CCR5 gene expression was maintained at initial transduction levels throughout the 3-week culture period. Colony forming assays (CFU) were performed to assess the effect of intrabody expression on hematopoietic development. No differences were seen in numbers of CFU and colony types when comparing parental, control vector, and intrabody expressing cells (n = 3). To determine the level of protection afforded by CCR5 intrabody expression, macrophages were exposed to JR-FL, a CCR5 using HIV-1, at 0.3 and 0.51 multiplicity of infection. Cultures were maintained for 15 – 23 days and evaluated for p24, a measure of viral replication, at 3 – 8 day intervals. At the end of culture it was found that macrophages expressing CCR5 intrabody generated 42-, 57-, 52-fold less p24 as compared to parental and control vector containing macrophages. Thus, CCR5 intrabody expression drastically decreased viral load. Therefore, reduction of CCR5 through vector delivery of intrabody genes to CD34+ cells should reduce the likelihood of infection of myeloid cells, their function as viral reservoirs, and has promising therapeutic potential. Currently, studies are underway to evaluate this modality of treatment in humanized NOD/SCID mice.

Alterations in mast cell function and survival following in vitro infection with human immunodeficiency viruses-1 through CXCR4

HIV-1 infection leads to a disease that attacks the central regulatory mechanisms of the immune response. As mucosal tissue is one of the primary sites infected with HIV in vivo, we examined the effects of HIV exposure on human mast cells, important components of mucosal defense. Using the human mast cell line, HMC-1, which expresses CXCR4 but not CCR5 on the cell surface, we found that several HIV-1 X4 tropic lab (IIIB, RF) and primary isolates but not R5 (BAL, ADA) isolates productively infected these cells. Furthermore, stem cell factor-dependent mast cells derived from primary fetal liver or cord blood cultures were also productively infected with both X4 and R5 HIV-1 strains. Infection was blocked at the level of viral entry using monoclonal antibodies to CXCR4 and CD4. Treatment of HMC-1 with TNF-α and TGF-β stimulated cell surface expression of CCR5 and up-regulated expression of both CCR5 and CXCR4 on primary mast cells, leading to increased susceptibility to both X4 and R5 viral isolates. HIV-1 infection also resulted in histamine release from these mast cells, most due in part to HIV-mediated cell death. These results demonstrate that X4 viruses can use CD4 and the CXCR4 receptor to infect mast cells, suggesting that mast cell–T cell interactions may contribute to HIV mediated immune dysfunction in the mucosa.

A CCR2-V64I polymorphism affects stability of CCR2A isoform.

A valine to isoleucine substitution at position 64 of CCR2 (CCR2-64I) is associated with a delay in progression to AIDS in HIV-1-infected individuals. The aim of the present study is to elucidate the molecular mechanism underlying the effect of this allele. We analysed the effect of the 64I substitution on levels of expression of CCR2A and CCR2B, two CCR2 isoforms produced by alternative splicing. Sendai virus vector was used to express CCR2 molecules. While CCR2B trafficked well to the cell surface, CCR2A, which differs from CCR2B only by the sequence of its C-terminal cytoplasmic tail, was detected predominantly in the cytoplasm. The level of expression of CCR2A-64I was significantly higher than that of CCR2A without the substitution. On the other hand, the 64I substitution did not affect levels of CCR2B expression. Pulse-chase experiments revealed that the 64I substitution increased the half-life of CCR2A in cells. When co-expressed with CCR5, CCR2A-64I interfered more severely with cell surface expression of CCR5 than did wild-type CCR2A. Furthermore, immunoprecipitation experiments showed that CCR2A co-precipitated with an immature form of CCR5. These results suggest that CCR2A binds to CCR5 in the cytoplasm and down-modulates its surface expression. We propose that the increased ability of CCR2A-64I to down-modulate CCR5 expression might be a possible cause of a delay in HIV-1 disease progression in patients with this allele.

Hemofiltrate CC chemokine 1[9-74] causes effective internalization of CCR5 and is a potent inhibitor of R5-tropic human immunodeficiency virus type 1 strains in primary T cells and macrophages.

Proteolytic processing of the abundant plasmatic human CC chemokine 1 (HCC-1) generates a truncated form, HCC-1[9-74], which is a potent agonist of CCR1, CCR3, and CCR5; promotes calcium influx and chemotaxis of T lymphoblasts, monocytes, and eosinophils; and inhibits infection by CCR5-tropic human immunodeficiency virus type 1 (HIV-1) isolates. In the present study we demonstrate that HCC-1[9-74] interacts with the second external loop of CCR5 and inhibits replication of CCR5-tropic HIV-1 strains in both primary T cells and monocyte-derived macrophages. Low concentrations of the chemokine, however, frequently enhanced the replication of CCR5-tropic HIV-1 isolates but not the replication of X4-tropic HIV-1 isolates. Only HCC-1[9-74] and HCC-1[10-74], but not other HCC-1 length variants, displayed potent anti-HIV-1 activities. Fluorescence-activated cell sorter analysis revealed that HCC-1[9-74] caused up to 75% down-regulation of CCR5 cell surface expression, whereas RANTES (regulated on activation, normal T-cell expressed and secreted) achieved a reduction of only about 40%. Studies performed with green fluorescent protein-tagged CCR5 confirmed that both HCC-1[9-74] and RANTES, but not full-length HCC-1, mediated specific internalization of the CCR5 HIV-1 entry cofactor. Our results demonstrate that the interaction with HCC-1[9-74] causes effective intracellular sequestration of CCR5, but they also indicate that the effect of HCC-1[9-74] on viral replication is subject to marked cell donor- and HIV-1 isolate-dependent variations.

Reduced Cell Surface Expression of CCR5 in CCR5Δ32 Heterozygotes Is Mediated by Gene Dosage, Rather Than by Receptor Sequestration

Macrophage tropic (M-tropic) human immunodeficiency virus (HIV) infection of primary human T cells and macrophages requires optimal cell surface expression of the chemokine receptor CCR5 in addition to CD4. Natural mutations of CCR5 that impair surface expression bestow in the homozygous state complete resistance to M-tropic HIV infection. ccr5Delta32 is the major prototype of such mutants. ccr5Delta32 heterozygosity is associated with delayed onset of AIDS and reduced risk of initial transmission, and this correlates with reduced levels of CCR5 and reduced infectability of CD4+ cells. In addition to gene dosage, sequestration of wild type (WT) CCR5 by mutant protein has been proposed as a mechanism to explain reduced surface expression of CCR5 in cells from ccr5Delta32 and CCR5-893(-) heterozygotes. However, here we demonstrate that a molar excess of ccr5Delta32 or related deletion mutants does not significantly impair the cell surface density of co-expressed WT receptor either in human epithelial cells or Jurkat T cells. Further, ligand-dependent signaling and M-tropic HIV usage of WT receptor are also unaffected. Nascent WT receptor does associate with ccr5Delta32 and related mutant proteins and with other unrelated CC and CXC chemokine receptors under transient labeling conditions. However, using confocal microscopy, we demonstrate that in the steady state, WT and truncated CCR5 proteins segregate into nonoverlapping subcellular compartments. These findings together with the observed and known variability in the cell surface density of CCR5 on quiescent PBLs lead us to conclude that reduced CCR5 gene dosage rather than receptor sequestration is the major determinant of reduced CCR5 expression in cells from ccr5Delta32 heterozygotes.