Trafficking machinery of NKT cells: shared and differential chemokine receptor expression among V alpha 24(+)V beta 11(+) NKT cell subsets with distinct cytokine-producing capacity

Natural killer T (NKT) cells are important regulators of the immune system, but their trafficking machinery, including expression of chemokine receptors, has been poorly defined. Unlike other conventional T-cell populations, we show that most NKT cells express receptors for extralymphoid tissue or inflammation-related chemokines (CCR2, CCR5, and CXCR3), while few NKT cells express lymphoid tissue-homing chemokine receptors (CCR7 and CXCR5). A population with homing potential for lymph nodes (L selectin(+) CCR7(+)) exists only within a small subset of CD4 NKT cells. We show differential expression of chemokine receptors among NKT cell subsets: CCR4 is mainly expressed by a high cytokine (interleukin-4/interleukin-2)-producing (CD4) NKT subset, while CCR1, CCR6, and CXCR6 are preferentially expressed by the low cytokine-producing CD8 and CD4(-)CD8(-) subsets. In line with this, TARC/CCL17 (a CCR4 ligand) induces preferential chemotaxis of the CD4 NKT subset, while chemotactic activities of LARC/CCL20 (a CCR6 ligand) and MIP-1 alpha/CCL3 (a CCR1 ligand) are focused on the CD8 and CD4(-)CD8(-) NKT cells. We conclude that, unlike conventional naive, memory, or effector T cells, the entire NKT cell population expresses nonlymphoid tissue homing chemokine receptors, yet NKT cell subsets differ considerably from each other by displaying distinct and reciprocal expression patterns of some chemokine receptors. Our results identify chemokine receptors that are potentially important for trafficking of human blood NKT cell subsets and reveal their function (cytokine production capacity)-dependent differential trafficking potentials.     

Expression and regulation of chemokine receptors in human natural killer cells

Using flow cytometric and RNase protection assays, this study examined the expression of chemokine receptors in nonactivated natural killer (NK) cells and compared this expression with NK cells activated with interleukin (IL)-2, which either adhered to plastic flasks (AD) or did not adhere (NA). None of the NK cell subsets expressed CXCR2, CXCR5, or CCR5. The major differences between these cells include increased expression of CXCR1, CCR1, CCR2, CCR4, CCR8, and CX(3)CR1 in AD when compared to NA or nonactivated NK cells. The chemotactic response to the CXC and CC chemokines correlated with the receptor expression except that all 3 populations responded to GRO-alpha, despite their lack of CXCR2 expression. Pretreatment of these cells with anti-CXCR2 did not inhibit the chemotactic response to GRO-alpha. In addition, nonactivated and NA cells responded to fractalkine, although they lack the expression of CX(3)CR1. This activity was not inhibited by anti-CX(3)CR1. Viral macrophage inflammatory protein (vMIP)-I, I-309, and TARC competed with the binding of (125)I-309 to AD cells with varying affinities. Transforming growth factor (TGF)-beta1 but not any other cytokine or chemokine examined including interferon (IFN)-gamma, MIP-3beta, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC) or I-309, up-regulated the expression of CXCR3 and CXCR4 on NK cell surface. This is correlated with increased chemotaxis of NK cells treated with TGF-beta1 toward stromal cell-derived factor (SDF)-1alpha and interferon-inducible protein-10 (IP-10). Messenger RNA for lymphotactin, RANTES, MIP-1alpha, and MIP-1beta, but not IP-10, monocyte chemotactic protein (MCP)-1, IL-8, or I-309 was expressed in all 3 NK cell subsets. Our results may have implications for the dissemination of NK cells at the sites of tumor growth or viral replication. (Blood. 2001;97:367-375)     

Oral biopsies from patients with orofacial granulomatosis with histology resembling Crohn's disease have a prominent Th1 environment

BACKGROUND: Orofacial granulomatosis (OFG) is an idiopathic inflammatory disorder of children and young adults whose clinical symptoms include swelling of the lips or face, mucosal nodularity (cobblestoning), mucosal tags, hyperplasia of the gingivae, and aphthous oral ulcers. Whether some OFG patients with clinical and histological characteristics resembling Crohn's disease (CD) are a special group (oral CD) or true CD patients with symptoms reaching all the way to the oral mucosa remains to be determined. METHODS: In this study oral biopsies from 10 patients with OFG were analyzed for the presence of T cells, T-cell subsets, B cells, and macrophages, as well as cytokines (IL-4, IL-10, IFN-gamma, IL-12, and TNF-alpha), chemokines (RANTES and MIP-1alpha), and chemokine receptors (CCR3, CCR5, and CXCR3). For comparison, oral tissues from 7 patients with other granulomatous diseases were included. RESULTS: Compared with the non-OFG group, the OFG group had raised levels of CD4(+) T cells, IFN-gamma, IL-10, and RANTES but reduced levels of CD68(+) macrophages outside the granulomas, whereas within the granulomas the levels of CD3(+) and CD4(+) T cells and of IFN-gamma were raised, but the levels of IL-4 were decreased. These data are indicative of a Th1 environment within the oral OFG tissues, which resembles that already observed in gut CD tissues. CONCLUSIONS: Therefore, it can be concluded that some OFG patients have both histopathological and immunopathological features that resemble those observed in CD patients.     

Differential expression of chemokine receptors on peripheral blood, synovial fluid, and synovial tissue monocytes/macrophages in rheumatoid arthritis

OBJECTIVE: Since it is likely that monocytes utilize chemokines to migrate to the rheumatoid arthritis (RA) joint, we investigated the expression of C-C chemokine receptors (CCR) 1-6 and C-X-C receptor 3 (CXCR3) in the peripheral blood (PB), synovial fluid (SF), and synovial tissue of patients with RA as well as in the PB of normal subjects. METHODS: We compared chemokine receptor expression on CD14+ monocytes from normal PB, RA PB, and RA SF using 2-color flow cytometry. Correlations with patient clinical data were determined. Chemokine and receptor expression were investigated in RA synovial tissue by immunohistochemistry and 2-color immunofluorescence to identify CD68+ macrophages. RESULTS: Most normal PB monocytes expressed CCR1 (87%) and CCR2 (84%), but not CCRs 3, 4, 5, or 6 or CXCR3. RA PB monocytes expressed CCR1 (56%) and CCR2 (76%), with significantly more expressing CCR3 (18%), CCR4 (38%), and CCR5 (17%) compared with normal PB monocytes. Significantly fewer SF monocytes from RA patients expressed CCR1 (17%), CCR2 (24%), and CCR4 (6%) while significantly more expressed CCR3 (35%) and CCR5 (47%) compared with RA and normal PB monocytes; CCR6 and CXCR3 were rarely detected. Clinically, the erythrocyte sedimentation rate was inversely correlated with the expression of CCR1 and CCR4 by RA PB, and CCR5 expression by RA SF was correlated with the SF white blood cell count. CCR1-, CCR2-, and CCR5-immunoreactive cells were found in RA synovial tissue and colocalized with CD68+ macrophages. RA synovial tissue RANTES (regulated upon activation, normally T cell expressed and secreted chemokine)- and monocyte chemoattractant protein 1-immunoreactive cells colocalized with CCR1 and CCR2, respectively, on serial sections. Macrophage inflammatory protein 1alpha (MIP-1alpha) was principally restricted to vascular endothelium, and MIP-1beta+ macrophages were found throughout the sections. CONCLUSION: Monocytes mainly express CCR1 and CCR2 in normal and RA PB, CCR3 and CCR5 in RA PB and RA SF, and CCR4 in RA PB. The differential expression of chemokine receptors suggests that certain receptors aid in monocyte recruitment from the circulation while others are important in monocyte retention in the joint.     

A low level of CD4+CD28+ T cells is an independent predictor of high mortality in human immunodeficiency virus type 1-infected patients

This study investigated coexpression of CD28, CD45RA, and CD45RO on CD4(+) and CD8(+) cells in 107 human immunodeficiency virus (HIV) type 1-infected patients, who were followed-up prospectively and were not treated with highly active antiretroviral therapy, and 65 control subjects. The most important novel finding was that a 50% reduction in CD4(+)CD28(+) cells predicted increased mortality (relative hazards [HR], 1.6; 95% confidence interval [CI], 1.0-2.6; P=.04), even after adjusting for the CD4(+) cell counts, virus load, beta(2)-microglobulin and hemoglobin levels, and HIV disease stage. Patients with progressed HIV infection had decreased concentrations of all studied cell subsets. Concerning the proportions of cells, only CD4(+)CD28(+), CD4(+)CD45RA(+), and CD8(+)CD45RO(+) cells decreased with HIV progression. Low proportions of CD4(+)CD45RA(+), CD8(+)CD45RA(+), and CD8(+)CD45RO(+) cells predicted mortality only in univariate but not in multivariate Cox analyses. If our results are confirmed in other studies, coexpression of CD28 on CD4(+) cells may be a useful marker to evaluate HIV progression.     

Selective lymphocyte chemokine receptor expression in the rheumatoid joint.

OBJECTIVE: In patients with rheumatoid arthritis (RA), chemokines and their receptors are important for lymphocyte trafficking into the inflamed joint. This study was undertaken to characterize the expression of chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CXCR3, and CX3CR1 in normal (NL) peripheral blood (PB), RA PB, and RA synovial fluid (SF). METHODS: Using flow cytometry, immunohistochemistry, and 2-color immunofluorescence, we defined the expression of chemokine receptors on CD3+ T lymphocytes in RA synovial tissue (ST), RA SF, RA PB, and NL PB. RESULTS: The percentage of CD3+ lymphocytes expressing CCR2, CCR4, CCR5, and CX3CR1 was significantly elevated in RA PB compared with that in NL PB, while the percentage of CD3+ lymphocytes expressing CCR5 was significantly enhanced in RA SF compared with that in NL and RA PB. In contrast, similar percentages of CD3+ lymphocytes in NL PB, RA PB, and RA SF expressed CCR6 and CXCR3. Immunohistochemistry of RA ST showed lymphocyte expression of CCR4, and 2-color immunofluorescence staining revealed RA ST CD3+ lymphocytes intensely immunoreactive for CXCR3, suggesting that these 2 receptors may be particularly important for CD3+ lymphocyte trafficking to the inflamed joint. In comparisons of chemokine receptor expression on naive (CD45RA+) and memory (CD45RO+) CD3+ lymphocytes, there were greater percentages of memory CD3+/CD4+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD4+ lymphocytes in RA PB and RA SF, and greater percentages of memory CD3+/CD8+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD8+ lymphocytes in RA SF, suggesting receptor up-regulation upon lymphocyte activation. In contrast, percentages of CD3+/CD8+ memory lymphocytes expressing CX3CR1 were significantly less than percentages of naive CD3+/CD8+ lymphocytes in RA PB, suggesting that this receptor may be down-regulated upon lymphocyte activation. A major difference between the RA PB and NL PB groups was significantly more CCR4+ memory leukocytes and memory CCR5+/ CD3+/CD8+ lymphocytes in RA PB than NL PB, further suggesting that these receptors may be particularly important for lymphocyte homing to the RA joint. CONCLUSION: These results identify CCR4, CCR5, CXCR3, and CX3CR1 as critical chemokine receptors in RA.     

Acceleration of idiopathic pneumonia syndrome (IPS) in the absence of donor MIP-1 alpha (CCL3) after allogeneic BMT in mice

Idiopathic pneumonia syndrome (IPS) is a significant cause of morbidity and mortality after bone marrow transplantation (BMT) in humans. We developed a murine IPS model in which lethal pre-BMT conditioning and allogeneic T cells results in the recruitment of host monocytes and then donor T cells into the lung by day 7 after BMT, concomitant with development of severe lung dysfunction. We reported the T cell-dependent production of the T cell-attracting chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the lungs of such recipient mice. We reasoned that MIP-1 alpha might be a critical mediator of IPS. Lethally conditioned mice received transplants of major histocompatibility complex-disparate marrow and either wild-type (MIP-1 alpha(+/+)) or knockout (MIP-1 alpha(-/-)) spleen cells. Recipients of MIP-1 alpha(-/-) cells exhibited accelerated mortality and a decrease in specific compliance that appeared earlier than in recipients of MIP-1 alpha(+/+) cells. Donor CD4(+) and CD8(+) T cell expansion was increased in the spleens of recipients of MIP-1 alpha(-/-) cells. Lungs of recipients of MIP-1 alpha(-/-) cells had earlier recruitment of both T-cell subsets by day 3 after BMT, concomitant with the influx of cells expressing the cytolysins granzymes A and B. Monocyte recruitment was not altered. Levels of inflammatory cytokines were not increased and levels of T cell-attracting chemokines were decreased. The level of the anti-inflammatory cytokine interleukin 13 (IL-13) was lower in the serum and lungs of recipients of MIP-1 alpha(-/-) cells, indicating a skewing toward a more inflammatory T helper cell type 1 (Th1) cytokine milieu. Donor-derived MIP-1 alpha may play a role in allogeneic-induced IPS by limiting aggressive expansion of CD4(+) and CD8(+) T cells.     

Characterization of subsets of CD4+ memory T cells reveals early branched pathways of T cell differentiation in humans

The pathways for differentiation of human CD4(+) T cells into functionally distinct subsets of memory cells in vivo are unknown. The identification of these subsets and pathways has clear implications for the design of vaccines and immune-targeted therapies. Here, we show that populations of apparently naive CD4(+) T cells express the chemokine receptors CXCR3 or CCR4 and demonstrate patterns of gene expression and functional responses characteristic of memory cells. The proliferation history and T cell receptor repertoire of these chemokine-receptor(+) cells suggest that they are very early memory CD4(+) T cells that have "rested down" before acquiring the phenotypes described for "central" or "effector" memory T cells. In addition, the chemokine-receptor(+) "naive" populations contain Th1 and Th2 cells, respectively, demonstrating that Th1/Th2 differentiation can occur very early in vivo in the absence of markers conventionally associated with memory cells. We localized ligands for CXCR3 and CCR4 to separate foci in T cell zones of tonsil, suggesting that the chemokine-receptor(+) subsets may be recruited and contribute to segregated, polarized microenvironments within lymphoid organs. Importantly, our data suggest that CD4(+) T cells do not differentiate according to a simple schema from naive --> CD45RO(+) noneffector/central memory --> effector/effector memory cells. Rather, developmental pathways branch early on to yield effector/memory populations that are highly heterogeneous and multifunctional and have the potential to become stable resting cells.     

Selective recruitment of polarized T cells expressing CCR5 and CXCR3 to the inflamed joints of children with juvenile idiopathic arthritis

OBJECTIVE: To study the expression of chemokine receptors CCR5 and CXCR3 and the Th1/Th2 cytokine balance in children with oligoarticular or polyarticular juvenile idiopathic arthritis (JIA). METHODS: Using 3-color immunofluorescence, we studied the expression of CCR5 and CXCR3 on, and T cell cytokine production by, paired samples of synovial fluid (SF) and peripheral blood (PB) T cells from 20 patients with oligoarticular- or polyarticular-onset JIA. Chemokine and cytokine phenotypes were also compared within the CD45RO+,CD3+ subsets. CCR5 genotypes were confirmed by polymerase chain reaction typing and sequencing. RESULTS: In the majority of samples, the number of T cells that were CCR5+ and CXCR3+ was higher in SF than in PB, and this difference was significant. One child was homozygous for the null A32 CCR5 allele; 4 others had lower expression of CXCR3 in SF than in blood. All samples showed strongly Th1-type cytokine production by synovial T cells compared with that by PB T cells. Both features were also markedly polarized within the synovial CD45RO+ subset compared with PB CD45RO+ T cells. CONCLUSION: The high expression of CCR5 and CXCR3 and high interferon-gamma:interleukin-4 ratios suggest a type 1 phenotype of SF T cells in JIA. The difference between CD45RO+ T cells from SF and from PB suggests that specific activation events have occurred in synovial T cells. We suggest that the highly activated, Th1-type phenotype of T cells within the chronically inflamed joints of children with JIA may reflect specific recruitment events that contribute to the polarization of these cells.     

Human CD8+ T cells store CXCR1 in a distinct intracellular compartment and up-regulate it rapidly to the cell surface upon activation

Activation and subsequent differentiation of naive CD8+ T cells lead to the development of memory subsets with distinct homing and effector capacities. On nonlymphoid homing subsets, expression of "inflammatory" chemokine receptors (such as CXCR3, CCR5, CX3CR1, and CXCR1) is believed to promote migration into sites of infection/inflammation. Here we show that CXCR1 can be up-regulated to the cell surface within minutes of activating human CD8+ T cells. No concurrent up-regulation of other inflammatory chemokine receptors was observed. Up-regulation of CXCR1 preferentially occurred on central memory CD8+ T cells-that is, cells with a lymph node homing phenotype-and was functionally relevant. Immunofluorescence microscopy showed CXCR1 to be present in intracellular vesicles that do not significantly colocalize with perforin, RANTES (regulated upon activation normal T cell expressed and secreted), or the lysosomal marker CD63. By contrast, partial colocalization with the Golgi marker GM130, the constitutive secretory pathway marker beta2-microglobulin, and the early endosome marker EEA1 was observed. Up-regulation of CXCR1 did not occur after T-cell receptor cross-linking. By contrast, supernatants from activated neutrophils, but not from monocytes or dendritic cells, induced its up-regulation. These results suggest that CD8+ T cells can rapidly adapt their homing properties by mobilizing CXCR1 from a distinct intracellular compartment.     

The characterization of chemokine production and chemokine receptor expression reveals possible functional cross-talks in AML blasts with monocytic differentiation

OBJECTIVE: The mechanisms regulating the trafficking of leukemic myeloid blasts are poorly understood. A differential expression of chemokines and chemokine receptors might account for some aspects of the pattern of invasion and accumulation of leukemic cells. We aimed at defining the pattern of chemokine and chemokine receptor expression of acute myeloid leukemia (AML) blasts in comparison with their putative normal cell counterparts. PATIENTS AND METHODS: Twenty-five cases of AML were analyzed by flow cytometry for the expression of several chemokine receptors and by RT-PCR for the expression of relevant chemokines. For selected chemokines, the production was confirmed by ELISA. AML blasts were also assessed for their migration capacity in response to autologous supernatants and recombinant chemokines. RESULTS: Undifferentiated AML (MO-M1 and some M2) express only CXCR4 on their surface and produce mainly inflammatory chemokines, resembling normal CD34+ progenitors. More differentiated AML (M4-M5 and some M2) have a more diversified receptor repertoire and, besides CXCR4, express the receptors for inflammatory chemokines and produce both constitutive and inflammatory chemokines, resembling resting and activated monocytes. In particular, M4-M5 blasts produce MCP-1 and MIP-3alpha and also express their specific receptors (CCR2 and, to a lesser extent, CCR6) and migrate in vitro in response to MCP-1 and MIP-3alpha and to their own supernatant. A significant correlation between extramedullary involvement and coexpression of MCP-1/CCR2 was found. CONCLUSIONS: These data suggest that chemokines and their receptors segregate within the different FAB subtypes and, by allowing cross-talk among members of the malignant clone, might help to explain some aspects of the pattern of invasion in AML with monocytic differentiation.     

Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages

CCR5 and CXCR4 are the major HIV-1 coreceptors for R5 and X4 HIV-1 strains, respectively, and a threshold number of CD4 and chemokine receptor molecules is required to support virus infection. Therefore, we used a quantitative fluorescence-activated cell sorting assay to determine the number of CD4, CCR5, and CXCR4 antibody-binding sites (ABS) on various T cell lines, T cell subsets, peripheral blood dendritic cells (PBDC), and monocyte-derived macrophages by using four-color fluorescence-activated cell sorting analysis on fresh whole blood. Receptor levels varied dramatically among the various subsets examined and typically varied from 2- to 5-fold between individuals. CCR5 was expressed at much higher levels in CD4+/CD45RO+/CD62L-true memory cells compared with CD4+/CD45RO+/CD62L+ cells. Fresh PBDC had the highest number of CCR5 ABS among the leukocyte subsets examined but had few CXCR4 ABS, affording a strategy for sort-purifying PBDC. In vitro maturation of PBDC resulted in median 3- and 41-fold increases in CCR5 and CXCR4 ABS, respectively. We found that macrophage colony-stimulating factor caused the greatest up-regulation of both CCR5 and CXCR4 on macrophage maturation (from approximately 5,000 to approximately 50, 000 ABS) whereas granulocyte-macrophage colony-stimulating factor caused a marked decrease of CXCR4 (from approximately 5,000 ABS to <500) while up-regulating CCR5 expression (from approximately 5,000 to approximately 20,000 ABS). Absolute ABS for CD4 and the major HIV-1 coreceptors serve as a more quantitative measure of cell surface expression, and we propose that this be used for future studies looking at the modulation of CD4 or chemokine receptor expression by cytokines, HIV-1 infection, or receptor polymorphisms.     

CCR5+ and CXCR3+ T cells are increased in multiple sclerosis and their ligands MIP-1α and IP-10 are expressed in demyelinating brain lesions

Multiple sclerosis (MS) is a T cell-dependent chronic inflammatory disease of the central nervous system. The role of chemokines in MS and its different stages is uncertain. Recent data suggest a bias in expression of chemokine receptors by Th1 vs. Th2 cells; human Th1 clones express CXCR3 and CCR5 and Th2 clones express CCR3 and CCR4. Chemokine receptors expressed by Th1 cells may be important in MS, as increased interferon-gamma (IFN-gamma) precedes clinical attacks, and IFN-gamma injection induces disease exacerbations. We found CXCR3(+) T cells increased in blood of relapsing-remitting MS, and both CCR5(+) and CXCR3(+) T cells increased in progressive MS compared with controls. Furthermore, peripheral blood CCR5(+) T cells secreted high levels of IFN-gamma. In the brain, the CCR5 ligand, MIP-1alpha, was strongly associated with microglia/macrophages, and the CXCR3 ligand, IP-10, was expressed by astrocytes in MS lesions but not unaffected white matter of control or MS subjects. Areas of plaque formation were infiltrated by CCR5-expressing and, to a lesser extent, CXCR3-expressing cells; Interleukin (IL)-18 and IFN-gamma were expressed in demyelinating lesions. No leukocyte expression of CCR3, CCR4, or six other chemokines, or anti-inflammatory cytokines IL-5, IL-10, IL-13, and transforming growth factor-beta was observed. Thus, chemokine receptor expression may be used for immunologic staging of MS and potentially for other chronic autoimmune/inflammatory processes such as rheumatoid arthritis, autoimmune diabetes, or chronic transplant rejection. Furthermore, these results provide a rationale for the use of agents that block CCR5 and/or CXCR3 as a therapeutic approach in the treatment of MS.     

T lymphopaenia in relation to body mass index and TNF-alpha in human obesity: adequate weight reduction can be corrective

OBJECTIVE: Although individuals with obesity are susceptible to infection, the underlying causes have not been fully identified. To investigate whether obesity affects immunity, we studied subjects with isolated obesity. DESIGN AND SUBJECTS: Thirty-four obese persons from our outpatient obesity clinic and 50 nonobese healthy control subjects were studied. The effects of weight reduction were evaluated in obese subjects on a very-low-energy diet. We examined blastogenic response, lymphocyte subsets, circulatory TNF-alpha, soluble TNF-alpha receptor 1, soluble TNF-alpha receptor 2, and in vitro TNF-alpha production in obesity. MEASUREMENTS: Lymphocyte subsets were analysed with flowcytometry. TNF-alpha and soluble TNF receptors levels were assayed using commercially available enzyme-linked immunosorbent assay kits. RESULTS: Blastogenic responses to phytohemagglutinin or concanavalin A of T cells, CD3(+), CD4(+), CD8(+), CD4(+)CD45RO(+), and TCR alpha beta T cells were significantly diminished in obese subjects. Strong negative correlations were observed between TCR alpha beta and body weight and BMI in obese subjects. Circulatory levels of TNF-alpha, soluble TNF-alpha receptors, and in vitro TNF-alpha production were significantly increased compared to nonobese subjects. In obese subjects, there were significant positive correlations between serum levels of TNF-alpha and waist-hip ratio, serum levels of soluble TNF-alpha receptor 1 and body weight, soluble TNF-alpha receptor 2 and BMI, and soluble TNF-alpha receptor 2 and waist-hip ratio. The T cell responses and previously reduced non-CD8 T cell subsets were increased significantly following weight reduction. CONCLUSIONS: Our results suggest that subsets of T cell populations and their function may be reduced in human obesity, and that this may be related, at least in part, to the elevated TNF-alpha production. Furthermore, this T cell dysfunction can be recovered by adequate weight reduction.     

Selective lymphocyte chemokine receptor expression in the rheumatoid joint

Objective

In patients with rheumatoid arthritis (RA), chemokines and their receptors are important for lymphocyte trafficking into the inflamed joint. This study was undertaken to characterize the expression of chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CXCR3, and CX₃CR1 in normal (NL) peripheral blood (PB), RA PB, and RA synovial fluid (SF).

Methods

Using flow cytometry, immunohistochemistry, and 2‐color immunofluorescence, we defined the expression of chemokine receptors on CD3+ T lymphocytes in RA synovial tissue (ST), RA SF, RA PB, and NL PB.

Results

The percentage of CD3+ lymphocytes expressing CCR2, CCR4, CCR5, and CX₃CR1 was significantly elevated in RA PB compared with that in NL PB, while the percentage of CD3+ lymphocytes expressing CCR5 was significantly enhanced in RA SF compared with that in NL and RA PB. In contrast, similar percentages of CD3+ lymphocytes in NL PB, RA PB, and RA SF expressed CCR6 and CXCR3. Immunohistochemistry of RA ST showed lymphocyte expression of CCR4, and 2‐color immunofluorescence staining revealed RA ST CD3+ lymphocytes intensely immunoreactive for CXCR3, suggesting that these 2 receptors may be particularly important for CD3+ lymphocyte trafficking to the inflamed joint. In comparisons of chemokine receptor expression on naive (CD45RA+) and memory (CD45RO+) CD3+ lymphocytes, there were greater percentages of memory CD3+/CD4+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD4+ lymphocytes in RA PB and RA SF, and greater percentages of memory CD3+/CD8+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD8+ lymphocytes in RA SF, suggesting receptor up‐regulation upon lymphocyte activation. In contrast, percentages of CD3+/CD8+ memory lymphocytes expressing CX₃CR1 were significantly less than percentages of naive CD3+/CD8+ lymphocytes in RA PB, suggesting that this receptor may be down‐regulated upon lymphocyte activation. A major difference between the RA PB and NL PB groups was significantly more CCR4+ memory leukocytes and memory CCR5+/CD3+/CD8+ lymphocytes in RA PB than NL PB, further suggesting that these receptors may be particularly important for lymphocyte homing to the RA joint.

Conclusion

These results identify CCR4, CCR5, CXCR3, and CX₃CR1 as critical chemokine receptors in RA.

     

T-lymphocyte production of macrophage inflammatory protein-1alpha is critical to the recruitment of CD8(+) T cells to the liver, lung, and spleen during graft-versus-host disease

To investigate the mechanism by which macrophage inflammatory protein-1alpha (MIP-1alpha) affects graft-versus-host disease (GVHD), the expression and function of MIP-1alpha in 2 murine models of GVHD were evaluated. In irradiated class I and class II disparate recipients, the expression of messenger RNA (mRNA) and protein for MIP-1alpha was significantly increased in GVHD target organs after transfer of allogeneic lymphocytes compared to syngeneic lymphocytes. When lymphocytes unable to make MIP-1alpha were transferred, there was a decrease in the production of MIP-1alpha in the liver, lung, and spleen of bm1 (B6.C-H2(bm1)/By) and bm12 (B6.C-H2(bm12)/KhEg) recipients compared to the transfer of wild-type splenocytes. At day 6 there was a 4-fold decrease in the number of transferred CD8(+) T cells in the lung and approximately a 2-fold decrease in the number of CD8(+) T cells in the liver and spleen in bm1 recipients after transfer of MIP-1alpha-deficient (MIP-1alpha(-/-)) splenocytes compared to wild-type (MIP-1alpha(+/+)) splenocytes. These differences persisted for 13 days after splenocyte transfer. In contrast, the number of donor CD4(+) T cells found in the liver and lung was significantly increased after the transfer of MIP-1alpha(-/-) compared to wild-type splenocytes in bm12 recipients from day 6 through day 10. Thus, the transfer of allogeneic T cells was associated with the enhanced expression of MIP-1alpha in both a class I and class II mismatch setting. However, the increased expression only led to enhanced recruitment of CD8(+), but not CD4(+), donor T cells. Production of MIP-1alpha by donor T cells is important in the occurrence of GVHD and functions in a tissue-dependent fashion.     

Production of CD8+ T cell nonlytic suppressive factors by CD28, CD38, and HLA-DR subpopulations

HIV infection may be modified by CD8(+) T cells by the production of nonlytic antiviral factors. To determine subpopulations that mediate nonlytic, antiviral activity, we examined the production of beta chemokines and of CD8 antiviral factor (CAF) by different subsets, using CD8(+) cells derived from 24 HIV-1-infected and 25 uninfected individuals. Subjects with CD8(+) cell counts greater than 200/microl produced increased levels of MIP-1alpha by CD8(+)CD28(+), CD8(+)CD38(-), and CD8(+)HLA-DR(+) subsets as compared with uninfected controls. CD8(+)CD38(-) cells produced higher levels of MIP-1beta and RANTES. CAF production was increased by CD8(+)CD38(+) and CD8(+)HLA-DR(+) cells of HIV-infected individuals as compared with uninfected controls. Chemokine production was increased by cells that do not express activation markers, whereas CAF activity was increased by cells expressing CD38 or HLA-DR. These findings shed light on CD8(+) T cell noncytotoxic antiviral factor production during HIV infection.     

C-C chemokine profile of cord blood mononuclear cells: selective defect in RANTES production

Three C-C chemokines inhibit human immunodeficiency virus (HIV) entry into macrophages: macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and regulated-upon activation, normal T-cell expressed and secreted (RANTES). We studied the ability of placental cord blood mononuclear cells (CBMC) to secrete these C-C chemokines in comparison to adult blood mononuclear cells (ABMC). CBMC had diminished ability to secrete RANTES, as determined by enzyme-linked immunosorbent assay. Secretion of MIP-1alpha and MIP-1beta were similar in CBMC and ABMC. Whereas MIP-1alpha and MIP-1beta secretion were comparable in monocytes and lymphocytes, RANTES was secreted primarily by lymphocytes. Flow cytometric analysis of RANTES expression showed diminished intracellular RANTES expression in cord blood lymphocytes (CBL) compared to adult (peripheral) blood lymphocytes (ABL). A subset analysis of RANTES-producing CBL and ABL demonstrated that RANTES was produced predominantly by CD8+/CD45RO+ cells. CBL had a reduced proportion of CD8+/CD45RO+ cells compared with ABL, which may account for the diminished RANTES secretion by CBMC. These results may be relevant to the pathogenesis of perinatal HIV infection. (Blood. 2000;95:715-718)     

CCR5 mediates specific migration of Toxoplasma gondii-primed CD8 lymphocytes to inflammatory intestinal epithelial cells

BACKGROUND & AIMS: Toxoplasma gondii, an obligate intracellular parasite, can invade intestinal epithelial cells and elicit a robust Th1 immune response. In this model of intestinal inflammation, CD8(+) intraepithelial lymphocytes (IELs) secrete transforming growth factor (TGF)-beta, which appears necessary for the maintenance of homeostasis in the intestine. However, the mechanism responsible for the IEL migration to the inflamed intestine is still unclear. METHODS: An in vitro coculture cell system was used to quantify the IEL attraction by an infected intestinal epithelial cell line (m-IC(cl2)). We used CCR5-deficient mice to determine which chemokine receptor-chemokine interaction could be responsible for the recruitment of antigen-specific CD8(+) IELs to the small intestine for the promotion of parasite clearance and host recovery. RESULTS: We observed increased expression of several chemokine receptors (CCR1, CCR2, CCR5, CXCR3) in the infected ileum. In particular, CCR5 expression was markedly increased in antigen-primed CD8(+) IELs. Experiments using recombinant chemokines as well as blocking antibodies showed that macrophage inflammatory protein (MIP)-1alpha and MIP-1beta were critical for their homing. CD8(+) IELs isolated from CCR5-deficient mice (CCR5-/-), despite their high production of TGF-beta and overexpression of activation markers, were impaired in their ability to migrate in vitro to the m-IC(cl2) monolayer or in vivo to the inflamed intestine after adoptive transfer. CONCLUSIONS: Our data emphasize the biologic role of CCR5 as an important component in the migration of intraepithelial CD8(+) T cells and the regulation of the inflammatory response following parasite infection.