Inhibition by IL-12 and IFN-alpha of I-309 and macrophage-derived chemokine production upon TCR triggering of human Th1 cells

Th1 and Th2 cells, which produce distinct sets of cytokines, differentially express several chemokine receptors that may regulate their tissue-specific localization. Although the expression pattern and regulation of chemokines are likely to play a critical role in many immunopathological processes, they remain largely unknown. Here, we investigated the requirements for Th1 and Th2 cells to produce the Th2 cell-attracting chemokines thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and I-309. TCR triggering of Th1 and Th2 cells leads to production of MDC and I-309 (CCR4 and CCR8 ligands, respectively), whereas TARC (CCR4 ligand) is selectively produced by Th2 cells. Secretion of these chemokines appears to be independent of endogenous production of IL-4 and IFN-gamma. IL-12 and IFN-alpha, cytokines that promote the differentiation of human Th1 cells, selectively inhibit secretion and mRNA expression of MDC and I-309 by Th1 cells. Suppression of I-309 secretion results in a decreased chemotactic effect on L1.2 cells transfected with human CCR8, indicating that IL-12 and IFN-alpha may inhibit the recruitment of CCR8-expressing cells such as Th2 cells. The inhibition of Th2 cell-attracting chemokines MDC and I-309 illustrates a novel mechanism by which IL-12 and IFN-alpha could promote and maintain an ongoing Th1 response.     

Chemokines, chemokine receptors and allergy

Chemokines are a group of cytokines that are responsible for the influx of blood cells, including T and B lymphocytes, monocytes, neutrophils, eosinophils and basophils, in allergic and other inflammatory conditions. They function as G protein-coupled chemotactic factors which also activate the cells with which they interact. Certain chemokines function within the afferent arm of the immune system, in which antigen is processed and antibody formation initiated, and others are active within the effector pathways of cellular immunity and late-phase allergic reactions. Th2 lymphocytes, which are critical for allergy, employ the CC chemokine receptors CCR4 and CCR8 with the ligands thymus- and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and I-309, respectively. The chemokine receptor CCR3 and ligands monocyte chemoattractant protein (MCP)-3, MCP-4, regulated upon activation normal T cell expressed and secreted (RANTES) and eotaxins I and II are of particular relevance for the recruitment and activation of eosinophils. Th1 reactions depend upon interferon gamma-induced CXC chemokines interferon- inducible protein (IP)-10, interferon-inducible T cell-alpha chemoattractant (iTAC) and monokine induced by interferon-gamma (MiG), which bind to chemokine receptor CXCR3.     

Skewing of cytotoxic activity and chemokine production,but not of chemokine receptor expression,in human type-1/-2 gamma delta T lymphocytes

Human Vgamma9/Vdelta2(+) T lymphocytes participate in the immune response against intracellular pathogens through the secretion of type-1 cytokines and chemokines and by killing of infected cells. Little is known of the effects by type-2 differentiation of gamma delta cells on these functions. Here, we report that bona fide naive cord blood-derived gamma delta lymphocytes expanded in vitro with the mycobacterial antigen isopentenyl pyrophosphate (IPP) can be differentiated as either type-1 or type-2 cells, in the presence of an appropriate cytokine milieu. Instead, peripheral gamma delta cells from PPD-negative healthy adults displayed a type-1 cytokine profile, i.e. IPP-stimulated secretion of IFN-gamma, but not of IL-4 and IL-10. Moreover, they released the macrophage inflammatory protein (MIP)-1beta, but not IL-8 nor the Th2 chemoattractants I-309 and TARC (thymus and activation-regulated chemokine). This cytokine profile was not significantly affected by in vitro culture in Th2 polarizing conditions. Only in one case out of seven were peripheral gamma delta cells fully differentiated to type-2 lymphocytes, characterized by sustained IL-4 and IL-10 production, along with secretion of substantial amounts of IL-8, I-309 and TARC. Type-2 gamma delta T lymphocytes preferentially expressed the co-stimulatory molecule CD30; conversely, no skewing in chemokine receptor expression was observed. Both polarized populations displayed high levels of CXCR3 in the absence of CCR3, CCR4 and CCR5. Finally, type-1, but not type-2, gamma delta T lymphocytes killed IPP-pulsed U937 cells and displayed elevated perforin content. Overall, our data suggest that type-2 differentiation of gamma delta T lymphocytes profoundly affects both their effector functions and their potential to recruit the appropriate leukocyte subsets to the sites of inflammation.     

Characterization of chemokines and chemokine receptors in two murine models of inflammatory bowel disease: IL-10-/- mice and Rag-2-/- mice reconstituted with CD4+CD45RBhigh T cells

We used quantitative PCR to investigate the expression of chemokines and chemokine receptors in two Th1-mediated murine models of inflammatory bowel disease (IBD). First, mRNA levels encoding the chemokines MIG, RANTES, lymphotactin, MIP-3alpha, TCA-3, TARC, MIP-3beta, LIX, MCP-1 and MIP-1beta and the receptors CCR4, CCR6 and CCR2 were significantly increased in chronically inflamed colons of IL-10-/- mice when compared with wildtype mice. Interestingly, reversal of colitis in IL-10-/- mice by anti-IL-12 mAb was accompanied by the inhibition in the expression of LIX, lymphotactin, MCP-1, MIG, MIP-3alpha, MIP-3beta, TCA-3, CCR2 and CCR4, whereas the increased mRNA levels of MIP-1beta, RANTES, TARC and CCR6 were unaffected. Second, to investigate which chemokines and receptors were up-regulated during the inductive phase of colitis, we employed the CD4+CD45RBhigh T cell transfer model. At 4 and 8 weeks after reconstitution of Rag-2-/- mice the mRNA levels of IP-10, MCP-1, MDC, MIG, TARC, RANTES, CCR4 and CCR5 were significantly increased prior to the appearance of macroscopic lesions. Other chemokines and chemokine receptors were clearly associated with the acute phase of the disease when lesions were evident. The sum of our studies with these two models identifies chemokines that are expressed at constant levels, irrespective of inflammatory responses, and those that are specifically associated with acute and/or chronic stages of Th1-driven colitis.     

Identification of the CC chemokines TARC and macrophage inflammatory protein-1β as novel functional ligands for the CCR8 receptor

Chemokines are key molecules in directing leukocyte migration toward sites of inflammation. We have previously cloned a putative CC chemokine receptor gene, TER1, whose expression is restricted to lymphoid tissues and cell lines. Recently, this receptor has been shown to signal in response to the human CC chemokine I-309 and thus it has been renamed CCR8 according to the current nomenclature. In the present study, we report the identification of the CC chemokines thymus and activation-regulated cytokine (TARC) and macrophage inflammatory protein-1β (MIP-1β) as CCR8 ligands, as they induce chemotaxis in CCR8 Jurkat stable transfectants. Furthermore, we have generated a polyclonal antiserum that is able to recognize the CCR8 molecule in transfectant lysates. The pattern of CCR8 mRNA expression and the functional effects exerted by its ligand suggest that the triggering of this receptor may regulate multiple functions including activation, migration and proliferation of lymphoid cells.     

Release of both CCR4-active and CXCR3-active chemokines during human allergic pulmonary late-phase reactions

BACKGROUND: Segmental antigen bronchoprovocation has long been used as a model to study allergic pulmonary inflammatory responses. Among the characteristics of the resulting cellular infiltrate is the preferential recruitment of TH2 lymphocytes. The mechanisms responsible for their selective recruitment remain unknown, but T(H)(2) cells preferentially express the chemokine receptors CCR4 and CCR8. OBJECTIVES: We tested the hypothesis that the chemokines thymus- and activation-regulated chemokine (TARC) (CCL17) and macrophage-derived chemokine (MDC) (CCL22), whose receptor is CCR4, and I-309 (CCL1), whose receptor is CCR8, would be released at sites of segmental allergen challenge. METHODS: Segmental allergen challenge with saline or allergen was performed in 10 adult allergic subjects with asthma, who were off medications. Bronchoalveolar lavage (BAL) was performed at both the saline- and allergen-challenged sites 20 hours after challenge. BAL fluids were analyzed for total cell counts and differentials, and supernatants were assayed by ELISA for levels of TARC, MDC, and I-309. As a control, the BAL fluids were also analyzed for levels of interferon-inducible protein 10 (IP-10) (CXCL10), an IFN-gamma-induced chemokine active on CXCR3, a chemokine receptor that is preferentially expressed on TH1 lymphocytes. RESULTS: Allergen challenge led to an approximately 6-fold increase in total leukocytes, including lymphocytes, compared with those seen at saline-challenged sites. At antigen-challenged sites, eosinophils predominated. Chemokine levels at control, saline-challenged sites were either below the detectable limit or low, with the predominant chemokine detected being IP-10. At antigen-challenged sites, levels of MDC, TARC, and IP-10 were all significantly increased compared with saline sites, each with a median of 486 to 1130 pg/mL detected. On the basis of a comparison with serum values, BAL chemokine levels at most antigen-challenged sites could not be accounted for by transudation from plasma. In contrast, levels of I-309 were extremely low or undetectable in all BAL and serum samples tested. Finally, BAL levels of MDC significantly correlated with those for TARC, but no significant correlations were found between levels of chemokine and any cell type. CONCLUSIONS: These data suggest that among the chemokines measured in this study, IP-10 is the predominant chemokine detected 20 hours after saline challenge, likely representing baseline production of a chemokine that favors TH1 cell recruitment. At antigen-challenged sites, levels of both CCR4 and CXCR3 active chemokines, but not CCR8 active chemokines, are markedly increased and are produced at levels that are likely to have biologic significance. Given the preferential accumulation of TH2 cells at these antigen-challenged sites, the increased production of CCR4-active chemokines might contribute to this response.     

A cytokine-to-chemokine axis between T lymphocytes and keratinocytes can favor Th1 cell accumulation in chronic inflammatory skin diseases

The recruitment of T cells into the skin is regulated by chemokines released by resident cells. In this study, we found that normal human keratinocytes activated with Th1-derived supernatant (sup) expressed early (6-12 h) IP-10/CXCL10, MCP-1/CCL2, IL-8/CXCL8, and I-309/CCL1 mRNAs and with slower kinetics (24-96 h), RANTES/CCL5 and MDC/CCL22 mRNAs. Upon stimulation with the Th1 sup, keratinocytes secreted high levels of RANTES, IP-10, MCP-1, and IL-8 and moderate levels of I-309 and MDC. Although much less efficiently, Th2 sup could also induce keratinocyte expression of IL-8, IP-10, RANTES, and MCP-1 but not of I-309 and MDC. TARC/CCL17 was not significantly induced by any stimuli. Sup from keratinocytes activated with Th1-derived cytokines elicited a strong migratory response of Th1 cells and a limited migration of Th2 cells, whereas sup from Th2-activated keratinocytes promoted a moderate migration of Th1 and Th2 lymphocytes. Thus, keratinocytes appear considerably more sensitive to Th1- than to Th2-derived lymphokines in terms of chemokine release and can support the preferential accumulation of Th1 lymphocytes in the skin.     

Switch in chemokine receptor expression upon TCR stimulation reveals novel homing potential for recently activated T cells.

When naive T lymphocytes are activated and differentiate into memory/effector cells, they down-regulate receptors for constitutive chemokines such as CXCR4 and CCR7 and acquire receptors for inflammatory chemokines such as CCR3, CCR5 and CXCR3, depending on the Th1/Th2 polarization. This switch in chemokine receptor usage leads to the acquisition of the capacity to migrate into inflamed tissues. Using RNase protection assays, staining with specific antibodies, and response to recombinant chemokines, we now show that following TCR stimulation, memory/effector T cells undergo a further and transient switch in receptor expression. CCR1, CCR2, CCR3, CCR5, CCR6 and CXCR3 are down-regulated within 6 h, while CCR7, CCR4, CCR8 and CXCR5 are up-regulated for 2 to 3 days. Up-regulation of CCR7 following TCR stimulation was observed also among resting peripheral blood T cells and required neither co-stimulation nor exogenous IL-2. On the other hand IL-2 down-regulated CXCR5, up-regulated CCR8 and facilitated the recovery of CCR3 and CCR5. Upon TCR stimulation, Th1 and Th2 cells produced comparable sets of chemokines, including RANTES, macrophage inflammatory protein-1beta, I-309, IL-8 and macrophage-derived chemokine, which may modulate surface chemokine receptors and contribute to cell recruitment at sites of antigenic recognition. Altogether these results show that following TCR stimulation effector/memory T cells transiently acquire responsiveness to constitutive chemokines. As a result, T cells that are activated in tissues may either recirculate to draining lymph nodes or migrate to nearby sites of organized ectopic lymphoid tissues.     

Distinct patterns and kinetics of chemokine production regulate dendritic cell function.

Dendritic cells (DC) have been showed to both produce and respond to chemokines. To understand how this may impact on DC function, we analyzed the kinetics of chemokine production and responsiveness during DC maturation. After stimulation with LPS, TNF-alpha or CD40 ligand, the inflammatory chemokines MIP-1alpha, MIP-1beta and IL-8 were produced rapidly and at high levels, but only for a few hours, while RANTES and MCP-1 were produced in a sustained fashion. The constitutive chemokines TARC, MDC and PARC were expressed in immature DC and were up-regulated following maturation, while ELC was produced only at late time points. Activated macrophages produced a similar spectrum of chemokines, but did not produce TARC and ELC. In maturing DC chemokine production had different impact on chemokine receptor function. While CCR1 and CCR5 were down-regulated by endogenous or exogenous chemokines, CCR7 levels gradually increased in maturing DC and showed a striking resistance to ligand-induced down-regulation, explaining how DC can sustain the response to SLC and ELC throughout the maturation process. The time-ordered production of inflammatory and constitutive chemokines provides DC with the capacity to self-regulate their migratory behavior as well as to recruit other cells for the afferent and efferent limb of the immune response.     

Chemotaxis of human tonsil B lymphocytes to CC chemokine receptor (CCR) 1, CCR2 and CCR4 ligands is restricted to non-germinal center cells

We have investigated the effects of nine CC chemokines, i.e. macrophage inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, MIP-3alpha/CCL20, MIP-5/CCL15, monocyte chemotactic protein (MCP)-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, eotaxin/CCL11 and macrophage-derived chemokine (MDC)/CCL22 on the locomotion of human tonsil B lymphocytes and their subsets. Upon isolation, B cells were poorly responsive, but, following short-term culture, they displayed statistically significant chemotactic responses (P < 0.001) to MIP-1alpha, MIP-5, MCP-1, MCP-2, MCP-3 and MDC. CC chemokine receptor (CCR) 1 to CCR6 were up-regulated after culture. MIP-1beta, MIP-3alpha and eotaxin did not stimulate B cell migration. Scattered information is available on B cell subset responses to chemokines. Therefore, we investigated the effects of MIP-1alpha, MIP-5, MCP-1, MCP-2, MCP-3 and MDC on the in vitro locomotion of non-germinal center (GC) (CD38(-)) and GC (CD38(+)) B cells. All chemokines enhanced significantly (P < 0.001) the migration of the former, but not of the latter, cells. CCR1, CCR2 and CCR4 were detected by flow cytometry on non-GC (i.e. naive and memory) B cells, whereas they were absent (CCR1 and CCR2) or poorly expressed (CCR4) on GC B cells.     

In vivo stability of human chemokine and chemokine receptor expression.

Cross-sectional analyses of human PBMC, plasma, and tissue have reported altered chemokine and/or chemokine receptor expression in several inflammatory diseases. Interpretation of such studies is difficult without data on the in vivo stability of such parameters. Using four color flow cytometry, we longitudinally followed CXCR3, CCR5 (Th1-associated), and CCR3 (Th2-associated) expression within CD4+/CD45RO+ and CD8+/CD45RO+ T cell populations in peripheral blood of healthy individuals over a 21 day period. In parallel, we quantified plasma levels of IP-10, Mig, eotaxin and TARC. Chemokine and receptor expression differed markedly between subjects but was highly stable, varying by <5% within individuals. Differences in chemokine receptor expression between subjects were markedly altered when quantified as absolute cell numbers rather than frequencies. Finally, CCR3 expression by CD4+/CD45RO+ T cells was positively correlated with plasma levels of its ligand, eotaxin, whereas strong negative correlations were evident between CXCR3 expression and IP-10 or Mig. These data demonstrate longitudinal stability of chemokine receptor and ligand expression among healthy individuals; reveal that both frequency and absolute cell count analysis is essential for accurate assessment of chemokine receptor expression; and identify inverse relationships between type 1 and type 2 immunity-associated receptors and their ligands in vivo.     

The chemokine receptor CCR8 mediates rescue from dexamethasone-induced apoptosis via an ERK-dependent pathway

Several chemokines have been shown to regulate cellular apoptosis following discrete stimuli. It was previously demonstrated that the CC chemokine CCL1 (I-309) rescues thymic lymphoma cells from apoptosis by unknown mechanisms. The aim of our study was to characterize the role of the CC chemokine receptor 8 (CCR8), the only described receptor for CCL1, in the rescue of murine thymic lymphoma cells and murine thymocytes from dexamethasone (dex)-induced apoptosis. We show here that the CCR8-restricted agonist Kaposi sarcoma-associated herpesvirus-encoded chemokine viral macrophage-inflammatory protein-1 (vMIP-1) rescues thymic lymphoma cells from dex-induced apoptosis, similar to CCL1, and that such rescue is extracellular-regulated kinase-dependent. Although it has been hypothesized that the rescuing effect of CCL1 from apoptosis could be CCR8-mediated, here, we formally demonstrate the role of such receptor as its selective antagonist encoded by the MC148 gene of molluscum contagiosum virus MC148/vMCC-I inhibits v-MIP-1- and CCL1-induced rescue activity. In addition, CCR8 ligands inhibit dex-induced apoptosis of murine thymocytes with potential implications for thymic selection.     

CC chemokine receptor 4 ligand production by bronchoalveolar lavage fluid cells in cigarette-smoke-associated acute eosinophilic pneumonia

We examined the production of macrophage-derived chemokine (MDC/CCL22) and thymus- and activation-regulated chemokine (TARC/CCL17) by bronchoalveolar lavage fluid (BALF) cells in cigarette-smoke-associated acute eosinophilic pneumonia (CS-AEP). The CC Chemokine Receptor 4 (CCR4) ligand levels in BALF from patients with CS-AEP were considerably higher than those in healthy volunteers and correlated well with Th2 cytokine levels. Interleukin-4 enhanced CCR4 ligand production. MDC expression was observed in CD68-positive cells from patients with CS-AEP and in healthy control smokers. In contrast, TARC expression in CD68- or CD1a-positive cells was detected only in CS-AEP. An in vivo cigarette smoke challenge test induced increases in CCR4 ligands in the BALF and in the cultured supernatant of BALF adherent cells. These results suggest that alveolar macrophages and dendritic cells contribute to the pathogenesis of CS-AEP by generating CCR4 ligands, probably in response to cigarette smoke.     

Increased responsiveness of murine eosinophils to MIP-1beta (CCL4) and TCA-3 (CCL1) is mediated by their specific receptors,CCR5 and CCR8

In the present study, we investigated the regulation of chemokine-mediated responses and receptor expression on eosinophils from mice. MIP-1alpha (CCL3) and eotaxin (CCL11) induced a significant and only partially overlapping intracellular calcium flux in antigen-elicited and peripheral blood eosinophils, and MCP-1 (CCL2), MDC (CCL22), MIP-1beta (CCL4), and TCA-3 (CCL1) did not. To demonstrate functional use of the specific receptors, we examined chemotactic responses. Peripheral blood eosinophils migrated toward MIP-1alpha (CCL3) and eotaxin (CCL11) but not MCP-1 (CCL2), MDC (CCL22), MIP-1beta (CCL4), and TCA-3 (CCL1). Antigen-elicited eosinophils migrated toward MIP-1alpha (CCL3) and eotaxin (CCL11), but also migrated in response to MIP-1beta (CCL4) and TCA-3 (CCL1), suggesting the up-regulation of additional chemokine receptors on antigen-elicited eosinophils. The up-regulation of the additional chemokine-receptor responses appeared to be in part because of cytokine activation, because TNF-alpha and/or IL-4 were able to up-regulate CCR1, -3, -5, and -8 mRNA expression in eosinophils as well as migration responses to the appropriate ligands. Using antibodies specific for CCR5 and CCR8, the chemotactic response to MIP-1beta and TCA-3, respectively, was reduced significantly. Finally, the expression of these new receptors appears to have an effect on activation and degranulation because MIP-1beta (CCL4) and TCA-3 (CCL1) induce significant levels of LTC4 from elicited eosinophils. These results suggest that eosinophils may up-regulate and use additional chemokine receptors during progression of inflammatory, allergic responses for migration and activation.     

Characterization of the phenotypic and lymphokine profile associated with strong CD8+ anti-HIV-1 suppressor activity (CASA)

A panel of 22 CD8+ T cell lines, with a broad range of CD8+ anti-HIV-1 suppressor activity (CASA) were generated from a single patient with HIV-1 infection. CD8+ T cell lines with either strong or weak CASA were examined and compared for cell surface and intracellular markers, constitutive chemokine and lymphokine mRNA levels and inducible lymphokine expression. Strong CASA significantly correlated with CD8+ T cell lines that highly coexpressed the molecule CD28+ (r=0.52, P=0.01) and Ki67+ (r=0.88, P=0.02), with strong CASA CD8+ T cell lines demonstrating significantly higher (P < 0.05) expression of CD8+CD28+ and CD8+Ki67+ compared to those with weak activity. No such correlations or findings were observed for the markers CD38, HLA-DR, CD57 or perforin. The Th1 cytokines were expressed at greater levels than the Th2 cytokines, with strong CASA significantly associated with an increased inducible level of IL-2 production (P=0.05). Constitutive RANTES, IP-10 and I-309 mRNA expression were significantly (P < 0.05) elevated in CD8+ T cell lines exhibiting strong CASA compared to those with weak CASA. There was no significant difference in the mRNA expression of the lymphokines IL-2, 4, 5, 8, 9, 10, 14, 15, or chemokines MIP-1alpha, MIP-1beta, MCP-1, and Ltn. Strong CASA was therefore associated with rapidly replicating CD8+ T cells of the phenotype CD8+CD28+Ki67+ that expressed greater levels of IL-2 and the ligands RANTES and I-309.