Role of the autocrine chemokines MIP-1alpha and MIP-1beta in the metastatic behavior of murine T cell lymphoma

The ESb-MP T-cell line is a highly malignant murine lymphoma, which preferentially metastasizes toward the kidney. This could be a result of the local production of monocyte chemoattractant protein-1 (MCP-1) and regulated on activation, normal T expressed and secreted (RANTES), which are chemotactic for ESb-MP cells. Here, we demonstrate that ESb-MP cells are already responsive to the chemotactic activity of macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta from 1 ng/ml onward. Moreover, upon stimulation with lipopolysaccharide (LPS) or virus, ESb-MP cells themselves produce significant amounts of MIP-1 ( approximately 200 ng/ml). Indeed, the major autocrine chemoattractants, isolated from ESb-MP cells, were intact MIP-1alpha and MIP-1beta. Pretreatment with LPS or addition of MIP-1 inhibited the in vitro migration of ESb-MP cells toward various chemokines. Moreover, compared with untreated lymphoma cells, LPS-treated cells produced significantly less metastasis in mice. The results represented here suggest that the role of chemokines in attracting tumor cells at secondary sites depends on a balance between autocrine-produced and tissue-derived chemokines. This delicate balance should be considered in the design of antichemokine strategies in different tumor types.     

Simian/human immunodeficiency virus(89.6) expressing the chemokine genes MIP-1alpha,RANTES, or lymphotactin

We constructed replication competent, attenuated, nef-deleted SHIV(89.6) that express the rhesus macaque chemokine genes MIP-1alpha, RANTES, or LTN from the nef region. The chemokine inserts were stable during several passages in CEMx174 cells and the viruses grew well in activated rhesus PBMC. Expression of virally encoded MIP-1alpha, RANTES, or LTN was detected in culture fluids from infected HOS CD4(+) CXCR4(+) cells, that were used because they have a low background production of these chemokines. The in vitro growth kinetics of all nef-deleted SHIV(89.6) were slower than the parental strain in both CEMx174 cells and rhesus PBMC. Rhesus macaques were susceptible to SHIV(89.6-MIP-1alpha), SHIV(89.6-RANTES), SHIV(89.6-LTN), and nef-deleted control SHIV(89.6-dLTN) infection via the intrarectal route using standard virus doses, and intact viruses were reisolated from infected animals throughout the interval of acute infection. SHIV expressing the chemokine genes MIP-1alpha, RANTES, or LTN may help determine the in vivo roles for these chemokines in modulating virus replication and disease.     

MIP-1 alpha and MIP-1 beta induction by dengue virus

Dengue virus (DV) infection can result in either a mild febrile illness known as dengue fever (DF) or a life-threatening disease called dengue hemorrhagic fever (DHF). DHF is more prevalent in patients undergoing secondary DV infection. This observation has led to the hypothesis that DHF may be the result of immune reactions to the secondary DV infection; an event termed immunopathology. Two cellular factors, MIP-1 alpha and MIP-1 beta, have been found to be induced by infection with DV. MIP-1 induction by DV infection was observed in a myelomonocytic cell line, as well as in peripheral blood mononuclear cells isolated from a dengue naive donor. MIP-1 induction was not due to factors secreted by infected cells. In fact, replication-competent virus was required to induce MIP-1. Evidence is also provided that MIP-1 genes are expressed in patients with dengue disease. It is hypothesized that these chemokines may have roles in the immunopathology of dengue infections and may contribute to fever and bone marrow suppression observed in patients with DV infections.     

Divergent regulation of HIV-1 replication in PBMC of infected individuals by CC chemokines: suppression by RANTES, MIP-1alpha, and MCP-3, and enhancement by MCP-1

We investigated the role of different CC chemokines, including regulated upon activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-lalpha (MIP-1alpha), monocyte chemotactic protein-1 (MCP-1), and MCP-3 on virus replication in cultures established from CD8+ T cell-depleted peripheral blood mononuclear cells (PBMC) of HIV-infected individuals that were either cocultivated with allogeneic T cell blasts (ATCB) of uninfected individuals or directly stimulated by mitogen plus interleukin-2. RANTES was the only chemokine that showed a clear-cut suppressive effect on HIV replication in both culture systems, although inhibitory effects were frequently also observed with MIP-1alpha, MCP-3, and, occasionally, with MCP-1. In contrast, MCP-1 frequently enhanced HIV production in most patients' cultures or cocultures that were characterized by secreting relatively low levels (<20 ng/mL) of MCP-1. When CD8-depleted PBMC of HIV+ individuals were cocultivated with ATCB of uninfected healthy donors, a positive correlation was observed between MCP-1 concentrations and the enhancement of HIV-1 replication occurring after depletion of CD8+ cells from donors' cells. Depletion of CD14+ cells (monocytes) from ATCB resulted in the down-regulation of virus replication during co-cultivation with CD8-depleted PBMC of infected individuals. Of interest, MCP-1 up-regulated HIV production in these CD14-depleted ATCB cocultures. Altogether these observations suggest that MCP-1 may represent an important factor enhancing HIV spreading, particularly in anatomical sites, such as the brain, where infection of macrophages and microglial cells plays a dominant role.     

Enumeration of interleukin-1 alpha and beta producing cells by flow cytometry.

A technique for intracytoplasmic immunofluorescence staining to detect and quantify human interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta) in CD4, CD8, and CD14 positive lymphoid cells is described. Mononuclear cells stimulated in vitro with PHA to produce IL-1, were fixed and made permeable to antibodies by sequential exposure to paraformaldehyde and the detergent n-octyl-glucoside. Cytoplasmic and surface staining of both forms of IL-1 were demonstrated by indirect fluorescence using IL-1 beta and IL-1 alpha specific mouse monoclonal antibodies and quantified with flow cytometry.     

Contrasting roles for RANTES and macrophage inflammatory protein-1 alpha (MIP-1 alpha) in a murine model of allergic peritonitis.

Cell accumulation and CC chemokine production were assessed in the peritoneal cavity of ovalbumin (OVA)-sensitized mice following antigen challenge. Intraperitoneal challenge with OVA induced a significant eosinophil influx from 6 h post-challenge with increased numbers persisting at 24 h. At 6 h there was also a marked presence of neutrophils. Messenger RNA expression and protein levels for the chemokines RANTES and MIP-1 alpha were measured in the cell pellets and supernatants, respectively, from peritoneal washes following OVA challenge. RANTES mRNA was detected from 2 h to 4 h following OVA injection, whereas mRNA for MIP-1 alpha was only detectable at 4 h. RANTES protein was first detected from 4 h after OVA injection and by 24 h the protein levels had increased further. Basal levels of MIP-1 alpha were detected in peritoneal washes. These levels peaked at 2 h after OVA challenge and rapidly declined to basal levels by 6 h. A functional role for the chemokines was assessed using neutralizing polyclonal antibodies. Co-injection of OVA with anti-RANTES antibodies resulted in a significant inhibition of eosinophil infiltration into the cavity at 6 h and 24 h (63% and 52% inhibition, respectively) without significantly influencing the number of neutrophils present. In contrast, injection of anti-MIP-1 alpha antibodies only inhibited neutrophil migration at the 6 h time point by 44% without significantly affecting the accumulation of eosinophils. These results demonstrate an important role for RANTES in mediating eosinophil influx in allergic inflammation and a contrasting role for MIP-1 alpha in mediating neutrophil recruitment.     

Altered intracellular expression of the chemokines MIP-1alpha,MIP-1beta and IL-8 by peripheral blood CD4+ and CD8+ T cells in mild allergic asthma

BACKGROUND: The ability of chemokines to regulate Th1 and Th2 responses suggests a role in the pathogenesis of atopic disorders such as allergic asthma where Th2 response dominance has been observed. Although the impact of allergic asthma on local chemokine production in the lung has been the subject of investigation, little is know about the influence of disease progression on peripheral chemokine production. We now report use of whole blood culture and flow cytometry to assess the influence of mild allergic asthma on peripheral T-cell chemokine expression. METHODS: Study participants included patients with mild allergic asthma (n = 7) and nonasthmatic controls (n = 7). Following in vitro stimulation of peripheral venous blood with phorbol 12-myristate acetate (PMA) and ionomycin, flow cytometry was used to estimate the percentage of CD4+ and CD8+ T cells producing a number of chemokines, including macrophage inflammatory proteins MIP-1alpha and MIP-1beta, RANTES (regulated on activation, T-cell expressed and secreted), monocytic chemotactic protein-1 (MCP)-1, and interleukin (IL)-8, or the cytokines interferon (IFN)-gamma and IL-4. Serum levels of MIP-1alpha, MIP-1beta, RANTES, MCP-1, IL-8, IFN-gamma and IL-4 were also assessed by quantitative ELISA. RESULTS: Intracellular expression of MIP-1beta by CD4+ and CD8+ T cells from allergic asthmatics was significantly reduced in comparison to that observed for nonasthmatics (median = 2.29% (1.75-3.50) vs 4.57% (3.38-6.64), P = 0.05; 14.20% (13.18-17.88) vs 44.10% (30.38-48.70), P = 0.01). Similarly, intracellular expression of MIP-1alpha by CD8+ T cells from allergic asthmatics was also significantly lower (3.67% (1.17-5.42) vs 17.10% (4.97-20.43), P = 0.05). Conversely, IL-8 expression by both CD4+ and CD8+ T cells from allergic asthmatics demonstrated significant enhancement (9.93% (7.77-11.28) vs 4.14% (3.61-7.11), P = 0.05; 8.40% (6.97-10.04) vs 4.98% (3.37-6.08), P = 0.05). Examination of intracellular IFN-gamma and IL-4 revealed no significant difference in the expression of either cytokine by CD4+ T-cells from allergic asthmatics and nonasthmatics. In contrast, expression of IFN-gamma was significantly reduced in CD8+ T-cells from allergic asthmatics (24.60% (21.08-32.50) vs 48.40% (41.50-55.28), P = 0.01). CONCLUSIONS: The occurrence in mild allergic asthma of peripheral T-cell chemokine expression suggestive of a diminished Th1 response, coinciding with marginal change in cytokine profiles indicative of a Th2 response bias, confirms the importance of chemokine involvement in the etiology of allergic asthma. The ability to use whole blood culture to estimate chemokine expression in T cell subsets may ultimately provide a practical means to evaluate disease status and to monitor early intervention therapies which target chemokines.     

A chimeric MIP-1alpha/RANTES protein demonstrates the use of different regions of the RANTES protein to bind and activate its receptors

Human RANTES (CCL5) and MIP-1alpha (CCL3) bind and activate several CC chemokine receptors. RANTES is a high-affinity ligand for CCR1 and CCR5, and it binds CCR3 with moderate affinity and CCR4 with low affinity. MIP-1alpha has similar binding characteristics to RANTES except that it does not bind to CCR3. Here we have generated a chimera of human MIP-1alpha and RANTES, called MIP/RANTES, consisting of the eight amino terminal residues of MIP-1alpha preceding the CC motif, and the remainder of the sequence is RANTES. The chimera is able to induce chemotaxis of human monocytes. MIP/RANTES has >100-fold reduction in binding to CCR1 and does not bind to CCR3 but retains full, functional binding to CCR5. It has equivalent affinity for CCR5 to MIP-1alpha and RANTES, binding with an IC(50) of 1.12 nM, and is able to mobilize calcium and induce endocytosis of CCR5 in PBMC in a manner equi-potent to RANTES. It also retains the ability to inhibit R5 using HIV-1 strains. Therefore, we conclude that the amino terminus of RANTES is not involved in CCR5 binding, but it is essential for CCR1 and CCR3.     

哮喘小鼠支气管MIP-1α、RANTES基因和蛋白的表达

目的 :研究巨噬细胞炎性蛋白 - 1α(MIP - 1α)、调节激活正常T细胞表达和分泌细胞因子 (RANTES)基因和蛋白在哮喘小鼠支气管中的表达情况。方法 :2 0只二级雄性BALB/C小鼠随机分为两组 :正常小鼠组 (A0 组 )和哮喘小鼠组 (B0 组 )。以卵清白蛋白 (OVA)致敏激发法复制小鼠哮喘模型 ,采用免疫组化法和原位杂交法测定两组小鼠支气管MIP - 1α、RANTES蛋白及基因的表达情况。结果 :免疫组化显示 ,B0 组支气管MIP - 1α、RANTES蛋白的表达均显著高于A0 组 (P <0 0 1) ,其主要表达细胞是上皮细胞 ;原位杂交亦显示 ,B0 组支气管MIP - 1α、RANTES基因的表达均显著高于A0 组 (P <0 0 1) ,其主要表达细胞是上皮细胞。结论 :本研究证明MIP - 1α和RANTES等在实验性哮喘时支气管中的高表达 ,上皮细胞是主要表达细胞。     

Binding of RANTES, MCP-1, MCP-3, and MIP-1alpha to cells in human skin.

Based on their ability to induce leukocyte chemotaxis and adhesion to endothelial cells (ECs), chemokines have been implicated in driving inflammatory leukocyte emigration. Recently, it was suggested that chemokines can accomplish their pro-emigratory role more effectively while being bound to the luminal surface of the ECs. Previously, such binding was demonstrated in situ in human skin for the prototype alpha-chemokine interleukin (IL)-8. Here we used an in situ binding assay to investigate the binding characteristics of several beta-chemokines in intact human skin. RANTES, MCP-1, and MCP-3 bound, similar to IL-8, in a specific saturable manner to the ECs of venules and small veins but not arteries or capillaries. RANTES inhibited MCP-1 and MCP-3 binding and vice versa, indicating that the EC binding sites are shared among these beta-chemokines; moreover, IL-8 and RANTES cross-competed for each other's binding, suggesting that the same chemokine binding sites are used by members of alpha- and beta-chemokine subfamilies. Conversely, MIP-1alpha did not bind to the ECs and did not compete for binding of RANTES. Analogous to IL-8, all of the tested beta-chemokines bound to the resident dermal cells. As a novel aspect of chemokine interaction with cells in normal skin, we observed specific, saturable binding of RANTES, MCP-1, and MCP-3 but not MIP-1alpha or IL-8 to the ECs of dermal afferent lymphatic vessels. RANTES, MCP-1, and MCP-3 also cross-competed for each other's binding to lymphatics, suggesting a common binding site with a novel chemokine binding profile. We suggest that the chemokine binding to the ECs of lymphatics may be involved in the process of leukocyte entry into the afferent lymphatic vessels.     

Augmented production of chemokines (monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis.

To determine the role of chemokines in the pathogenesis of systemic sclerosis (SSc), we examined serum levels, spontaneous production by peripheral blood mononuclear cells (PBMC), and histological distribution in the affected skin, of MCP-1, MIP-1alpha and MIP-1beta in SSc patients. Serum levels of these chemokines were examined by ELISA in 58 patients with SSc and 20 normal controls. The levels of these chemokines in culture supernatants from PBMC were also measured by ELISA. Serum levels and spontaneous production levels by PBMC of MCP-1, MIP-1alpha, and MIP-1beta were significantly elevated in patients with SSc compared with normal controls. Elevated serum levels of MCP-1 and MIP-1alpha significantly correlated with the presence of pulmonary fibrosis. MCP-1 expression in the skin of SSc was immunohistochemically examined using anti-MCP-1 MoAb. MCP-1 was strongly expressed in the epidermis, inflammatory mononuclear cells, and vascular endothelial cells in the sclerotic skin of SSc patients, but not expressed in any control skin. Furthermore, the MCP-1 expression in inflammatory mononuclear cells and endothelial cells significantly correlated with earlier onset of SSc. Thus, MCP-1, MIP-1alpha and MIP-1beta may be involved in the disease process, possibly by augmenting leucocyte migration into the affected tissues in SSc. Furthermore, MCP-1 and MIP-1alpha may play an important role in the development of pulmonary fibrosis in SSc.     

17β-雌二醇对子宫内膜间质细胞体外分泌趋化因子IL-8、MIP-1α、RANTES和I-309的影响

目的：探讨17β-雌二醇对子宫内膜间质细胞分泌IL-8、MIP-1α、RANTES和I-309的调控，以解析雌激素在子宫内膜异位症发生发展中的作用机制。方法：用不同浓度17β-雌二醇处理子宫内膜间质细胞48小时；此后选用10^-9mol／L 17β-雌二醇处理子宫内膜间质细胞12、24、36和48小时，ELISA法分析刺激前后培养上清中IL-8、MIP-1α、RANTES和I-309分泌水平。结果：子宫内膜间质细胞体外高水平自然分泌IL-8，17β-雌二醇能抑制IL-8的分泌，呈剂量依赖性。RNATES和MIP-1α分泌水平较IL-8低，经雌激素刺激子宫内膜间质细胞后MIP-1α表现出与IL-8相同的分泌方式，但对RANTES分泌无明显影响。子宫内膜间质细胞经雌激素刺激前后均未检测到I-309。结论：雌二醇可能参与子宫内膜白细胞和单核巨噬细胞的募集，但不是异位灶中上述趋化因子高表达的原因。     

RANTES, MCP-1 chemokines and factors describing rheumatoid arthritis

The MCP-1/CCL2 as well as RANTES/CCL5 chemokines are potent chemoattractants involved in immunoregulatory and inflammatory processes of rheumatoid arthritis. Recent studies demonstrated elevated levels of MCP-1 and RANTES in plasma, synovial fluid, and the synovial tissue of patients with RA. To examine the relationship among MCP-1 and RANTES single nucleotide polymorphisms and circulating levels and rheumatoid arthritis (RA), a total of 156 RA patients and 125 controls were recruited into the study. An association of -855 C/G MCP-1 polymorphism to IgM RF within the RA patients was observed. The lowest circulating levels of RANTES were observed in the AA variant of RANTES -403 G/A polymorphism. Furthermore, an association of -403 AA variant to circulating levels of IL-15 and IL-10 was found. No associations of factors describing rheumatoid arthritis (RFs, ANA, anti-CCP-positive/negative, DAS 28 score and number of swollen joints) with MCP-1 levels, genotype distribution, allelic frequencies and/or frequencies of haplotypes composed of all three studied polymorphisms in promoter region of MCP-1, and RANTES polymorphism were observed. We conclude that the RANTES promoter polymorphism is associated to circulating levels of RANTES, IL15 and IL10. However, our findings suggest that polymorphisms in the MCP-1 and RANTES gene promoters do not contribute significantly to the interindividual RA susceptibility and/or severity in Caucasians.     

Lipopolysaccharide from an Escherichia coli htrB msbB mutant induces high levels of MIP-1 alpha and MIP-1 beta secretion without inducing TNF-alpha and IL-1 beta

OBJECTIVE: To identify a lipopolysaccharide (LPS) that retains the capacity to induce beta-chemokine secretion without the concomitant activation of pyrogenic cytokines. METHODS: LPS was extracted from strain MLK986 (mLPS), an htrB1::Tn10, msbB::ocam mutant of Escherichia coli that is defective for lipid A synthesis, and from wild-type parent E coli strains, W3110 (wtLPS). The capacity of these LPS preparations to induce tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and macrophage inflammatory proteins 1 alpha (MIP-1 alpha) and MIP-1 beta was assessed using a human peripheral blood mononuclear cell (PBMC) activation assay. RESULTS: Stimulation of PBMCs with mLPS did not induce measurable levels of pyrogenic cytokines TNF-alpha and IL-1 beta, whereas wtLPS induced high levels of these cytokines. Furthermore, mLPS antagonized the induction of TNF-alpha secretion by wtLPS. Nonetheless, mLPS retained a discrete agonist activity that induced MIP-1 alpha and MIP-1 beta secretion by PBMCs. This latter agonist activity appears to be unique to mLPS, since two previously documented LPS antagonists, Rhodobacter sphaeroides diphosphoryl lipid A and synthetic lipid IVA, did not induce MIP-1 alpha and MIP-1 beta secretion. Furthermore, synthetic lipid IVA was an antagonist of MIP-1 alpha and MIP-1 beta induction by mLPS. CONCLUSION: These results show that mLPS exhibits a novel bipartite activity, being an effective antagonist of TNF-alpha induction by wtLPS, while paradoxically being an agonist of MIP-1 alpha and MIP-1 beta secretion.     

PGE2抑制LPS诱导腹腔巨噬细胞产生MIP-1α和MIP-1β

目的:在离体情况下,研究PGE2能否抑制脂多糖(LPS)诱导腹腔巨噬细胞MIP1α和MIP1β的产生。方法:制备腹腔巨噬细胞,应用夹心酶联免疫分析法(SandwichELISA)检测MIP1α和MIP1β的浓度,并应用实时逆转录聚合酶链反应(realtimeRTPCR)法检测MIP1α和MIP1βmRNA的表达。结果:PGE2抑制腹腔巨噬细胞产生MIP1α和MIP1β,呈时间依赖性和浓度依赖性,抑制发生在mRNA和蛋白质水平,PGE2对巨噬细胞的抑制作用通过EP4和EP2介导。结论:PGE2能抑制MIP1α和MIP1β产生而调节免疫反应。     

Cryptococcus neoformans induces macrophage inflammatory protein 1alpha (MIP-1alpha) and MIP-1beta in human microglia: role of specific antibody and soluble capsular polysaccharide

We characterized the expression of the beta-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES by primary human microglia after exposure to Cryptococcus neoformans. In the absence of specific antibody, C. neoformans failed to elicit a chemokine response, while in the presence of specific antibody, microglia produced MIP-1alpha and MIP-1beta in amounts comparable to those induced by lipopolysaccharide. RANTES was also induced but at much lower levels. In addition to MIP-1alpha and MIP-1beta mRNA, we observed a robust induction of monocyte chemoattractant protein 1 and interleukin-8 mRNA following incubation of microglia with opsonized C. neoformans. In contrast, cryptococcal polysaccharide did not induce a chemokine response even when specific antibody was present and inhibited the MIP-1alpha induction associated with antibody-mediated phagocytosis of C. neoformans. The role of the Fc receptor in the observed chemokine induction was explored in several experiments. Treatment of microglia with cytochalasin D inhibited internalization of C. neoformans but did not affect MIP-1alpha induction. In contrast, treatment with herbimycin A, a tyrosine kinase inhibitor, inhibited MIP-1alpha induction. Microglia stimulated with immobilized murine immunoglobulin also produced MIP-1alpha and RANTES (MIP-1alpha > RANTES). Our results show that microglia produce several chemokines when stimulated by C. neoformans in the presence of specific antibody and that this process is likely to be mediated by Fc receptor activation. This response can be down-regulated by cryptococcal capsular polysaccharide. These findings suggest a mechanism by which C. neoformans infections fail to induce strong inflammatory responses in patients with cryptococcal meningoencephalitis and have important implications for antibody therapy.     

MIP-3alpha and MIP-1alpha rapidly mobilize dendritic cell precursors into the peripheral blood

Acquisition of dendritic cells (DCs) or DC precursors in vitro is critical for DC-based immunotherapy. We reported previously that administration of MIP-1alpha mobilized a population of F4/80(-)B220(-)CD11c+ DC precursors into peripheral blood by the expression of CCR1 and CCR5. In this study, we identified a new subset of CCR6+CCR1(-)CCR5(-)B220(-)CD11c(+) cells in MIP-1alpha-administered mice. When cultured with GM-CSF, IL-4, and TNF-alpha, these cells differentiated into mature DCs, possessing the typical morphologic characteristics, phenotypes, and antigen-presenting function (termed CCR6+ DC precursors). Although it did not directly drive the CCR6+ DC precursors, MIP-1alpha could recruit a population of F4/80+CD11c(-) monocyte/macrophage-producing MIP-3alpha in the peripheral blood to mobilize a CCR6+ DC precursor subset of B220(-)CD11c+ DC precursors. Importantly, exogenous administration of MIP-3alpha significantly enhanced MIP-1alpha-induced mobilization of DC precursors. Moreover, these MIP-3alpha- and MIP-1alpha-mobilized DC precursors could be prepared for a DC vaccine capable of eliciting CTL responses to tumor cells, leading to tumor rejection in vitro and in vivo. Taken together, this study further demonstrates the mechanism of DC precursor mobilization induced by MIP-1alpha; that is, besides mobilizing DC precursors with CCR1 and CCR5 expressions, MIP-1alpha recruited F4/80+CD11c(-) monocyte/macrophage-producing MIP-3alpha, which finally mobilized the CCR6+ DC precursor subset to amplify the B220(-)CD11c+ DC precursor population. Furthermore, combined administration of MIP-3alpha and MIP-1alpha may be an efficient strategy for collecting a large number of DCs appropriate for immunotherapy.     

RANTES and MIP-1beta mRNA expression in human peripheral blood mononuclear cells: transcript quantification using NASBA technology

The importance of chemokines in the immune response, as well as in a range of specific disease states, is becoming increasingly apparent. The role of CC- (or beta-) chemokines and their receptors in the pathology and mechanisms of HIV-1 infection has served to intensify interest in these factors. Although the functionality of these factors resides in their protein forms, assays for the detection and quantification of these protein factors in clinical samples are not readily available. Consequently, we designed NASBA-based assays for the quantification of the mRNA encoding two members of the CC-chemokine family: RANTES and MIP-1beta. The NASBA-based assays are extremely sensitive, accurate, and reproducible across a dynamic range of at least four orders of magnitude. Inter-assay performance is comparable to intra-assay performance. We applied these methods to the analysis of normal human PBMC and PBMC from HIV-1 infected individuals. Although MIP-1beta mRNA levels are higher than RANTES levels in both populations, RANTES levels in HIV-1+ patients are higher than in normal individuals. The utility of these assays in longitudinal studies of specific subpopulations of cells, as well as their potential use in clinical diagnostics, is discussed.