Molecular mimicry of cytokines and cytokine receptors is a strategy used by poxviruses and herpesviruses to modulate host immunity. The human cytomegalovirus (HCMV) UL144 gene, situated in the UL/b' region of the viral genome, has amino-acid sequence similarity to members of the tumour necrosis factor receptor superfamily. We report that UL144 is a potent activator of NFkappaB-induced transcription in a TRAF6-dependent manner. This NFkappaB activation enhances expression of the chemokine CCL22 through the NFkappaB responsive elements found in its promoter. In contrast to the clinical HCMV isolates, extensively passaged laboratory strains lack the UL/b' region and hence do not encode UL144. Consistent with this, infection with viruses that carry UL/b' causes NFkappaB activation and CCL22 expression, a phenotype that is not observed after infections with strains lacking the UL/b' region. Moreover, knockdown of UL144, TRAF6 or NFkappaB by specific siRNA in infections with UL144-encoding HCMV prevents the activation of CCL22 expression normally observed after infection with UL/b' positive HCMV. Upregulation of CCL22, which attracts Th2 and regulatory T cells, may help HCMV evade immune surveillance.

Increasing evidence suggests that the development of pulmonary fibrosis is a Th2-mediated process. We hypothesized that the CC chemokines that are associated with a Th2 profile (CCL17 and CCL22) have an important role in the development of pulmonary fibrosis. We measured CCL17 and CCL22 during the pathogenesis of bleomycin-induced pulmonary fibrosis. We found that both CCL17 and CCL22 were significantly elevated through day 20 as compared with control mice. Peak expression of CCL22 preceded the peak levels of CCL17, as measured by real-time quantitative PCR. CCR4 is the receptor for CCL17 and CCL22 therefore, to further characterize the role of CCL17 and CCL22, we measured CCR4 mRNA in lung tissue of bleomycin-treated mice by real-time quantitative PCR. CCR4 was significantly elevated in bleomycin-treated mice as compared with control mice. Immunolocalization demonstrated that CCR4 was expressed predominantly on macrophages. Neutralization of CCL17, but not CCL22, led to a reduction in pulmonary fibrosis. Immunolocalization of bleomycin-treated lung tissue and human idiopathic pulmonary fibrosis tissue specimens showed that epithelial cells expressed CCL17. These findings demonstrate a central role for Th2 chemokines and the macrophage in the pathogenesis of pulmonary fibrosis and are further support for the role of a Th2 phenotype in the pathogenesis of pulmonary fibrosis.

The presence of regulatory T cells (Treg) has been described in a large panel of solid tumors. However, their impact on tumor progression differs according to the tumor type analyzed. We recently obtained evidence in breast carcinoma that Treg localized within lymphoid aggregates, but not in the tumor bed, have a negative impact on patients' survival. Moreover, we showed selective Treg recruitment through CCR4/CCL22 in the lymphoid aggregates upon contact with dendritic cells (DC), where they became strongly and selectively activated (ICOS(high)) and block conventional T-cell response. Here, we discuss the meaning and potential implication of these novel findings.

Matrix metalloproteinases (MMPs) are a large family of endopeptidases that proteolytically degrade extracellular matrix. Many different cells produce MMP-9, and levels have been shown to be up-regulated in patients with allergic asthma. The aim of this study was to investigate the in vivo role of MMP-9 during allergen-induced airway inflammation. Acute allergic pulmonary eosinophilia was established in MMP-9 knockout (KO) and wild-type (WT) control mice by sensitization and challenge with OVA. Cell recruitment was significantly increased in both bronchoalveolar lavage (BAL) and lung tissue compartments in MMP-9 KO mice compared with WT mice. This heightened cell recruitment was primarily due to increased eosinophils and Th2 cells in the BAL and lung tissue of MMP-9 KO mice in comparison with WT controls. Moreover, levels of the Th2 cytokines, IL-4 and IL-13, and the chemokines eotaxin/CCL11 and macrophage-derived chemokine/CCL22 were substantially increased in MMP-9 KO mice compared with WT after OVA challenge. Resolution of eosinophilia was similar between MMP-9 KO and WT mice, but Th2 cells persisted in BAL and lungs of MMP-9 KO mice for longer than in WT mice. Our results indicate that MMP-9 is critically involved in the recruitment of eosinophils and Th2 cells to the lung following allergen challenge, and suggest that MMP-9 plays a role in the development of Th2 responses to allergen.

T-helper (Th) 2 cytokines are thought to mediate most features of allergic inflammation in atopic asthma. However, it remains unclear whether chemokine pathways direct selective recruitment of Th2 cells to the airways during human allergic responses. Bronchoalveolar lavage (BAL) was performed in 15 nonsmoking mild atopic asthmatics before and 24 h after a fibreoptic segmental allergen challenge, and chemokines related to T-cell recruitment were assayed by ELISA. The Th2-related C-C chemokine (CCR)4 ligands, macrophage-derived chemokine/C-C chemokine ligand (CCL)22 and thymus and activation-regulated chemokine/CCL17, were increased in BAL after challenge. These chemokines correlated significantly with lymphocyte numbers and with interleukin (IL)-5 and IL-13 in post-challenge BAL. In contrast, two out of three putative Th1-related chemokines did not change. There were no alterations in monokine induced by interferon (IFN)-gamma/CXC chemokine ligand (CXCL)9 or macrophage inflammatory protein-1alpha/CCL3; whereas a significant increase in IFN-induced protein-10kDa/CXCL10 was observed, which did not correlate with the T-cell influx. In peripheral mononuclear cells from atopic donors, CCL22 and CCL17 were induced by IL-4 and IL-13, further supporting the relationship between CCL22/CCL17 and Th2 cytokines. Finally, CCL22 was able to trigger actin polymerisation in peripheral CD4+ T-cells expressing CCR4. Thus, C-C chemokine receptor 4 ligands are up-regulated in the airways of atopic asthmatics following allergen exposure, contribute to the T-cell influx to the airways and are closely related to the Th2-cytokine response.

Natalizumab exerts impressive therapeutic effects in patients with multiple sclerosis (MS). The proposed main mode of action is reducing transmigration of leukocytes into the CNS, but other immunological effects may also be operative. Cytokines and chemokines are involved in the regulation of inflammatory responses and may reflect the disease process in MS. The objective of this study was to evaluate the effects of natalizumab treatment on cytokine and chemokine profiles systemically and intrathecally in multiple sclerosis. We used luminex to analyse a panel of cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma, GM-CSF) and chemokines (CXCL9, CXCL10, CXCL11, CCL17, CCL22) in blood and cerebrospinal fluid (CSF) from 31 patients with relapsing MS before and after one year of natalizumab treatment. There was a marked decline in CSF levels of cytokines and chemokines, thus including pro-inflammatory cytokines (IL-1beta, IL-6 and IL-8) as well as chemokines associated with both Th1 (CXCL9, CXCL10, CXCL11) and Th2 (CCL22). Circulating plasma levels of some cytokines (GM-CSF, TNF-alpha, IL-6 and IL-10) also decreased after one year of treatment. This is the first study to show that natalizumab treatment is associated with a global decline in cytokine and chemokine levels at a protein level. This finding was most pronounced in CSF, in line with the reduced transmigration of cells into CNS, whereas reduction in plasma levels indicates other possible mechanisms of natalizumab treatment.

There is increasing evidence of T cell dysfunction in B cell chronic lymphocytic leukaemia (B-CLL) which may contribute to the aetiology and progress of the disease. An absolute CD8+ lymphocytosis correlates with disease progression and low expression of CD4 and CD8 (as found in autoimmune disease) is seen with abnormal expression of other surface molecules. Although the expression of T cell surface activation markers, CD25 and CD152, may be increased on culture in B-CLL serum, response to the common mitogens, PHA and PWM, is reduced. This and the excess of CD8 cells may explain partly the variable cooperation of T cells with B cell production of immunoglobulin in B-CLL. In the context of T cell cross-talk with antigen presenting cells, B-CLL B cells are poor antigen presenters. But the T cells themselves have significant abnormalities of expression of the many antigens and ligands necessary for this process. In particular, they exhibit variable expression of the low affinity and non-specific adhesion molecules LFA-1 and ICAM-1, variable, clonally restricted and skewed expression of the TCR repertoire (implying repeated antigenic stimulation possibly by CLL antigens), reduced CD28 and CD152 expression (implying impairment of ability to start or stop an immune response) and reduced IL2 and CD25 (IL2 R) expression (critical for positive feed-back in maintenance and expansion of the T cell response to antigen presentation). Although the production of IL2 and other cytokines by the T cell in B-CLL may be impaired, production of the anti-apoptotic cytokine IL4 is not and there may be a unique and expanded subset of CD8/CD30 cells capable of releasing IL4. The relationship of this T cell subset to the malignant B cell in vivo is unknown. However, T cells which are CD4+/CD152+/CCR4+ migrate selectively in vitro in response to the chemokine CCL22 (specific for the receptor CCR4) produced by the malignant B cells and are always seen amongst the malignant cells in bone marrow and lymph nodes from B-CLL patients. Other abnormalities of cytokine secretion are described. These findings suggest that the T cell in B-CLL may be unable to start, maintain and complete an immune response to the malignant B cell and other antigens and may be involved directly in sustaining the tumour. However, autologous tumour specific cytotoxicity has been shown in vitro and T cells which recognise tumour-derived heavy chain fragments circulate in vivo. If adoptive immunotherapy of any nature is to succeed in B-CLL, manipulation to optimise these CTL responses is needed to overcome the profound and variable T cell dysfunction in this disease.

Chemokines are critical molecules in leukocyte trafficking, promoting site-specific migration to various tissues. The chemokine receptor CCR4 has recently been associated with skin-homing T cells. In view of the potential importance of CCR4 in skin homing of T cells, we investigated the expression pattern of CCR4 and its ligands TARC/CCL17 and MDC/CCL22 in the peripheral blood and skin of patients with cutaneous T cell lymphoma, a putative malignancy of the skin-homing T cells. In this study we analyzed the pattern of coexpression of the skin-homing molecules cutaneous lymphocyte antigen (CLA) and CCR4 in the blood and skin of patients with cutaneous T cell lymphoma. In the blood of cutaneous T cell lymphoma patients with peripheral blood involvement we found significantly increased percentages of T cells displaying the skin-homing phenotype (CLA+CCR4+) compared with healthy individuals. T cells expressing CLA and CCR4 were also found at high levels in cutaneous T cell lymphoma lesions along with abundant expression of the two CCR4 ligands TARC/CCL17 and MDC/CCL22. These data may explain, in part, why these T cells accumulate in the skin, a diagnostic feature of cutaneous T cell lymphomas.

BACKGROUND

Chemokines and their receptors are important elements for the selective attraction and activation of various subsets of leukocytes. Expression of CXCR3 ligands, such as monokine induced by IFN-gamma (Mig) leads to preferential Th1 recruitment, whereas CCR4 ligands, thymus and activation regulated chemokine (TARC) or macrophage derived chemokine (MDC), mediate preferential Th2 recruitment. Although atopic dermatitis (AD) has been shown to be a Th2-type disease, recent studies have revealed that Th1-type cytokines, such as IFN-gamma, especially in chronic skin lesions, play important roles in pathogenesis of AD.

OBJECTIVE

The purpose of this study was to investigate serum levels of Th2 chemokines TARC and MDC and a Th1 chemokine Mig in the same samples from patients with AD and their clinical correlation.

METHODS

Serum chemokine levels in patients with AD (n = 55), contact dermatitis (CD; n = 15), and normal controls (n = 30) were examined by ELISA.

RESULTS

Serum levels of TARC and MDC in AD patients and CD patients were significantly higher than those found in normal controls. Serum levels of these chemokines were similar for AD patients and CD patients. Furthermore, these levels correlated positively with disease severity, total IgE levels, and peripheral eosinophilia in AD patients. Serum Mig levels in AD patients and CD patients were significantly higher than those in control subjects. However, serum Mig levels were significantly elevated in CD patients relative to AD patients. Furthermore, serum Mig levels correlated positively with levels of both TARC and MDC in AD patients.

CONCLUSIONS

These results suggest that both Th2 and Th1 chemokines may play roles in the development of AD.

Although much has been learned recently of the mechanisms by which the differentiation of osteoclasts is induced, less is known of the factors that regulate their migration and localization, and their interactions with other bone cells. In related cell types, chemokines play a major role in these processes. We therefore systematically tested the expression of RNA for chemokines and their receptors by osteoclasts. Because bone is the natural substrate for osteoclasts and may influence osteoclast behavior, we also tested expression on bone slices. Quantitative RT-PCR using real-time analysis with SYBR Green was therefore performed on RNA isolated from bone marrow cells after incubation with macrophage-colony stimulating factor (M-CSF) with/without receptor-activator of NFkappaB ligand (RANKL), on plastic or bone. We found that RANKL induced expression of CCL9/MIP-1gamma to levels comparable to that of tartrate-resistant acid phosphatase (TRAP), a major specialized product of osteoclasts. CCL22/MDC, CXCL13/BLC/BCA-1, and CCL25/TECK were also induced. The dominant chemokine receptor expressed by osteoclasts was CCR1, followed by CCR3 and CX3CR1. Several receptors expressed on macrophages and associated with inflammatory responses, including CCR2 and CCR5, were down-regulated by RANKL. CCL9, which acts through CCR1, stimulated cytoplasmic motility and polarization in osteoclasts, identical to that previously observed in response to CCL3/MIP-1alpha, which also acts through CCR1 and is chemotactic for osteoclasts. These results identify CCL9 and its receptor CCR1 as the major chemokine and receptor species expressed by osteoclasts, and suggest a crucial role for CCL9 in the regulation of bone resorption.

In breast carcinomas, patient survival seems to be negatively affected by the recruitment of regulatory T cells (T(reg)) within lymphoid aggregates by CCL22. However, the mechanisms underpinning this process, which may be of broader significance in solid tumors, have yet to be described. In this study, we determined how CCL22 production is controlled in tumor cells. In human breast carcinoma cell lines, CCL22 was secreted at low basal levels that were strongly increased in response to inflammatory signals [TNF-α, IFN-γ, and interleukin (IL)-1β], contrasting with CCL17. Primary breast tumors and CD45(+) infiltrating immune cells appeared to cooperate in driving CCL22 secretion, as shown clearly in cocultures of breast tumor cell lines and peripheral blood mononuclear cells (PBMC) or their supernatants. We determined that monocyte-derived IL-1β and TNF-α are key players as monocyte depletion or neutralization of these cytokines attenuated secretion of CCL22. However, when purified monocytes were used, exogenous human IFN-γ was also required to generate this response suggesting a role for IFN-γ-producing cells within PBMCs. In this setting, we found that human IFN-γ could be replaced by the addition of (i) IL-2 or K562-activated natural killer (NK) cells or (ii) resting NK cells in the presence of anti-MHC class I antibody. Taken together, our results show a dialogue between NK and tumor cells leading to IFN-γ secretion, which in turn associates with monocyte-derived IL-1β and TNF-α to drive production of CCL22 by tumor cells and subsequent recruitment of T(reg). As one validation of this conclusion in primary breast tumors, we showed that NK cells and macrophages tend to colocalize within tumors. In summary, our findings suggest that at early times during tumorigenesis, the detection of tumor cells by innate effectors (monocytes and NK cells) imposes a selection for CCL22 secretion that recruits T(reg) to evade this early antitumor immune response.

Adult T cell leukemia/lymphoma (ATLL) is an aggressive malignancy caused by human T cell lymphotropic virus type-I (HTLV-I) without curative treatment at present. To illuminate the pathogenesis of ATLL we performed whole transcriptome sequencing of purified ATLL patient samples and discovered recurrent somatic mutations in CCR4, encoding CC chemokine receptor 4. CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330, or Y331 in the carboxy terminus. Functionally, the CCR4-Q330 nonsense isoform was gain-of-function because it increased cell migration toward the CCR4 ligands CCL17 and CCL22, in part by impairing receptor internalization. This mutant enhanced PI(3) kinase/AKT activation after receptor engagement by CCL22 in ATLL cells and conferred a growth advantage in long-term in vitro cultures. These findings implicate somatic gain-of-function CCR4 mutations in the pathogenesis of ATLL and suggest that inhibition of CCR4 signaling might have therapeutic potential in this refractory malignancy.

We have recently reported that mice deficient in the myeloid Src-family tyrosine kinases Hck, Fgr, and Lyn (Src triple knockout [TKO]) had augmented innate lung clearance of Pneumocystis murina that correlated with a higher ability of alveolar macrophages (AMs) from these mice to kill P. murina. In this article, we show that despite possessing enhanced killing, AMs from naive Src TKO mice did not demonstrate enhanced inflammatory responses to P. murina. We subsequently discovered that both AMs and lungs from P. murina-infected Src TKO mice expressed significantly greater levels of the M2a markers RELM-α and Arg1, and the M2a-associated chemokines CCL17 and CCL22 than did wild-type mice. IL-4 and IL-13, the primary cytokines that promote M2a polarization, were not differentially produced in the lungs between wild-type and Src TKO mice. P. murina infection in Src TKO mice resulted in enhanced lung production of the novel IL-1 family cytokine IL-33. Immunohistochemical analysis of IL-33 in lung tissue revealed localization predominantly in the nucleus of alveolar epithelial cells. We further demonstrate that experimental polarization of naive AMs to M2a resulted in more efficient killing of P. murina compared with untreated AMs, which was further enhanced by the addition of IL-33. Administration of IL-33 to C57BL/6 mice increased lung RELM-α and CCL17 levels, and enhanced clearance of P. murina, despite having no effect on the cellular composition of the lungs. Collectively, these results indicate that M2a AMs are potent effector cells against P. murina. Furthermore, enhancing M2a polarization may be an adjunctive therapy for the treatment of Pneumocystis.

Even though melanoma is considered to be one of the most immunogenic solid tumors, handling its development remains a challenge. The basis for such escape from antitumor immune control has not yet been documented. Plasmacytoid dendritic cells (pDC) are emerging as crucial but still enigmatic cells in cancer. In melanoma, the function of tumor-infiltrating pDCs remains poorly explored. We investigated the pathophysiologic role of pDCs in melanoma, both ex vivo from a large cohort of melanoma patients and in vivo in melanoma-bearing humanized mice. pDCs were found in high proportions in cutaneous melanoma and tumor-draining lymph nodes, yet associated with poor clinical outcome. We showed that pDCs migrating to the tumor microenvironment displayed particular features, subsequently promoting proinflammatory Th2 and regulatory immune profiles through OX40L and ICOSL expression. Elevated frequencies of interleukin (IL)-5-, IL-13- and IL-10-producing T cells in patients with melanoma correlated with high proportions of OX40L- and ICOSL-expressing pDCs. Strikingly TARC/CCL17, MDC/CCL22, and MMP-2 found in the melanoma microenvironment were associated with pDC accumulation, OX40L and ICOSL modulation, and/or early relapse. Thus, melanoma actively exploits pDC plasticity to promote its progression. By identifying novel insights into the mechanism of hijacking of immunity by melanoma, our study exposes potential for new therapeutic opportunities.

The expression of distinct chemokines within the asthmatic lung suggests that specific regulatory mechanisms may mediate various stages of asthmatic disease. Global transcript expression profiling was used to define the spectrum and kinetics of chemokine involvement in an experimental murine model of asthma. Seventeen chemokines were induced in the lungs of allergen-inoculated mice, as compared with saline-treated mice. Two (CXCL13 and CCL9) of the 17 identified chemokines have not previously been associated with allergic airway disease. Seven (7 of 17; CCL2, CCL7, CCL9, CCL11, CXCL1, CXCL5, CXCL10) of the allergen-induced chemokines were induced early after allergen challenge and remained induced throughout the experimental period. Three chemokines (CXCL2, CCL3, and CCL17) were induced only during the early phase of the inflammatory response after the initial allergen challenge, while seven chemokines (CCL6, CCL8, CCL12, CCL22, CXCL9, CXCL12, and CXCL13) were increased only after a second allergen exposure. Unexpectedly, expression of only three chemokines, CCL11, CCL17, and CCL22, was STAT6 dependent, and many of the identified chemokines were overexpressed in STAT6-deficient mice, providing an explanation for the enhanced neutrophilic inflammation seen in these mice. Notably, IFN-gamma and STAT1 were shown to contribute to the induction of two STAT6-independent chemokines, CXCL9 and CXCL10. Taken together, these results show that only a select panel of chemokines (those targeting Th2 cells and eosinophils) is positively regulated by STAT6; instead, many of the allergen-induced chemokines are negatively regulated by STAT6. Collectively, we demonstrate that allergen-induced inflammation involves coordinate regulation by STAT1, STAT6, and IFN-gamma.

BACKGROUND

Severe atopic dermatitis (AD) has a high unmet need for effective and safe therapeutics. In early-phase trials, dupilumab, a fully human mAb targeting IL-4 receptor α, markedly improved disease activity, but the effect of IL-4/IL-13 blockade on AD at the molecular level has not been characterized.

OBJECTIVE

We sought to evaluate dupilumab modulation of the AD molecular signature.

METHODS

We performed transcriptomic analyses of pretreatment and posttreatment skin biopsy specimens from patients with moderate-to-severe AD treated weekly with 150 or 300 mg of dupilumab or placebo.

RESULTS

Exacerbation of the AD transcriptome was observed in placebo-treated patients. Dupilumab improved the AD signature in a dose-dependent manner. Expression of genes upregulated in AD lesions decreased in patients treated with dupilumab by 26% (95% CI, 21% to 32%) and 65% (95% CI, 60% to 71%) for treatment with 150 and 300 mg, respectively. Genes downregulated in AD lesions increased by 21% (95% CI, 16% to 27%) and 32% (95% CI, 26% to 37%) with dupilumab (150 and 300 mg, respectively). The molecular changes paralleled improvements in clinical scores. A dupilumab treatment signature of 821 probes (>2-fold change, P < .05) significantly modulated in the 300-mg dupilumab group at week 4 compared with baseline was identified in this sample set. Significant (P < .05) decreases in mRNA expression of genes related to hyperplasia (K16 and MKI67), T cells, and dendritic cells (CD1b and CD1c) and potent inhibition of TH2-associated chemokines (CCL17, CCL18, CCL22, and CCL26) were noted without significant modulation of TH1-associated genes (IFNG).

CONCLUSIONS

This is the first report showing rapid improvement of the AD molecular signature with targeted anti-IL-4 receptor α therapy. These data suggest that IL-4 and IL-13 drive a complex, TH2-centered inflammatory axis in patients with AD.

Emerging evidence suggests a role for eosinophils in immune regulation of T cells. Thus, we sought to determine whether human eosinophils may exert their effect via differential generation of Th1 and Th2 chemokines depending on cytokines in their microenvironment and, if so, to establish the conditions under which these chemokines are produced. Eosinophils cultured with TNF-alpha plus IL-4 had increased mRNA expression and protein secretion of the Th2-type chemokines, CCL17 (thymus and activation-regulated chemokine) and CCL22 (macrophage-derived chemokine). Conversely, the Th1-type chemokines, CXCL9 (monokine induced by IFN-gamma) and CXCL10 (IFN-gamma-inducible protein-10), were expressed after stimulation with TNF-alpha plus IFN-gamma. Addition of TNF-alpha appeared to be essential for IFN-gamma-induced release of Th1-type chemokines and significantly enhanced IL-4-induced Th2-type chemokines. Inhibition of NF-kappaB completely blocked the production of both Th1 and Th2 chemokines. Activation of NF-kappaB, STAT6, and STAT1 was induced in eosinophils by TNF-alpha, IL-4, and IFN-gamma, respectively. However, there was no evidence for enhancement of these signaling events when eosinophils were stimulated with the combination of TNF-alpha plus IL-4 or TNF-alpha plus IFN-gamma. Thus, independently activated signaling cascades appear to lead to activation of NF-kappaB, STAT1, and STAT6, which may then cooperate at the promoter level to increase gene transcription. Our data demonstrate that TNF-alpha is a vital component for eosinophil chemokine generation and that, depending on the cytokines present in their microenvironment, eosinophils can promote either a Th2 or a Th1 immune response, supporting an immunoregulatory role for eosinophils.

Breast cancer is a leading cause of neoplasia-associated death in women worldwide. Regulatory T (Treg) and Th17 cells are enriched within some tumors, but the role these cells play in invasive ductal carcinoma (IDC) of the breast is unknown. We show that CD25(+) CD4(+) T cells from PBMCs and tumor express high levels of Foxp3, GITR, CTLA-4, and CD103, indicating that tumor-infiltrating Treg cells are functional and possibly recruited by CCL22. Additionally, we observed upregulation of Th17-related molecules (IL-17A, RORC, and CCR6) and IL-17A produced by tumor-infiltrating CD4(+) and CD8(+) T lymphocytes. The angiogenic factors CXCL8, MMP-2, MMP-9, and vascular endothelial growth factor detected within the tumor are possibly induced by IL-17 and indicative of poor disease prognosis. Treg and Th17 cells were synchronically increased in IDC patients, with positive correlation between Foxp3, IL-17A, and RORC expression, and associated with tumor aggressiveness. Therefore, Treg and Th17 cells can affect disease progression by Treg-cell-mediated suppression of the effector T-cell response, as indicated by a decrease in the proliferation of T cells isolated from PBMCs of IDC patients and induction of angiogenic factors by IL-17-producing Th17. The understanding of regulation of the Treg/Th17 axis may result in novel perspectives for the control of invasive tumors.

Previous studies in our laboratory have shown that platelets are essential for the migration of eosinophils into the lungs of allergic mice, and that this is dependent on the functional expression of platelet P-selectin. We sought to investigate whether the same is true for nonallergic, acute inflammatory stimuli administered to distinct anatomic compartments. Neutrophil trafficking was induced in two models, namely zymosan-induced peritonitis and LPS-induced lung inflammation, and the platelet dependence of these responses investigated utilizing mice rendered thrombocytopenic. The relative contribution of selectins was also investigated. The results presented herein clearly show that platelet depletion (>90%) significantly inhibits neutrophil recruitment in both models. In addition, we show that P-selectin glycoprotein ligand-1, but not P-selectin, is essential for neutrophil recruitment in mice in vivo, thus suggesting the existence of different regulatory mechanisms for the recruitment of leukocyte subsets in response to allergic and nonallergic stimuli. Further studies in human blood demonstrate that low-dose prothrombotic and pro-inflammatory stimuli (CCL17 or CCL22) synergize to induce platelet and neutrophil activation, as well as the formation of platelet-neutrophil conjugates. We conclude that adhesion between platelets and neutrophils in vivo is an important event in acute inflammatory responses. Targeting this interaction may be a successful strategy for inflammatory conditions where current therapy fails to provide adequate treatment.

Langerhans cells (LC) are epidermal dendritic cells capable, in several experimental systems, of Ag-presentation for stimulation of cell-mediated immunity. LC have been considered to play a key role in initiation of cutaneous immune responses. Additionally, administration of donor T cells to bone marrow chimeric mice with persistent host LC, but not mice whose LC have been replaced by donor cells, exhibit marked skin graft-vs-host disease, demonstrating that LC can trigger graft-vs-host disease. However, experiments with transgenic mice in which regulatory elements from human langerin were used to drive expression of diphtheria toxin, resulting in absence of LC, suggest that LC may serve to down-regulate cutaneous immunity. LC are associated with nerves containing the neuropeptide calcitonin gene-related peptide (CGRP), and CGRP inhibits LC Ag-presentation in several models including presentation to a Th1 clone. We now report that CGRP enhances LC function for stimulation of Th2 responses. CGRP exposure enhanced LC Ag presentation to a Th2 clone. Upon presentation of chicken OVA by LC to T cells from DO11.10 chicken OVA TCR transgenic mice, pretreatment with CGRP resulted in increased IL-4 production and decreased IFN-gamma production. CGRP also inhibited stimulated production of the Th1 chemokines CXCL9 and CXCL10 but induced production of the Th2 chemokines CCL17 and CCL22 by a dendritic cell line and by freshly obtained LC. Changes in production of these chemokines correlated with the effect of CGRP on mRNA levels for these factors. Exposure of LC to nerve-derived CGRP in situ may polarize them toward favoring Th2-type immunity.

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.

OBJECTIVE

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.

Adjuvants are substances that enhance immune responses and thus improve the efficacy of vaccination. Few adjuvants are available for use in humans, and the one that is most commonly used (alum) often induces suboptimal immunity for protection against many pathogens. There is thus an obvious need to develop new and improved adjuvants. We have therefore taken an approach to adjuvant discovery that uses in silico modeling and structure-based drug-design. As proof-of-principle we chose to target the interaction of the chemokines CCL22 and CCL17 with their receptor CCR4. CCR4 was posited as an adjuvant target based on its expression on CD4(+)CD25(+) regulatory T cells (Tregs), which negatively regulate immune responses induced by dendritic cells (DC), whereas CCL17 and CCL22 are chemotactic agents produced by DC, which are crucial in promoting contact between DC and CCR4(+) T cells. Molecules identified by virtual screening and molecular docking as CCR4 antagonists were able to block CCL22- and CCL17-mediated recruitment of human Tregs and Th2 cells. Furthermore, CCR4 antagonists enhanced DC-mediated human CD4(+) T cell proliferation in an in vitro immune response model and amplified cellular and humoral immune responses in vivo in experimental models when injected in combination with either Modified Vaccinia Ankara expressing Ag85A from Mycobacterium tuberculosis (MVA85A) or recombinant hepatitis B virus surface antigen (rHBsAg) vaccines. The significant adjuvant activity observed provides good evidence supporting our hypothesis that CCR4 is a viable target for rational adjuvant design.

BACKGROUND

Studies with monocyte-derived macrophages (MDMs) and animal models have suggested a role for alternatively activated (M2) macrophages in asthmatic inflammation, but in vivo evidence for this phenotype in human asthma is lacking.

OBJECTIVE

To characterize the phenotype of lung macrophages from asthmatic patients in relation to disease severity and treatment.

METHODS

M2 biomarkers were first identified by using MDMs exposed to T(H)2 cytokines and then used to phenotype sputum and bronchoalveolar lavage (BAL) macrophages from 12 healthy control subjects, 12 patients with mild asthma, and 14 patients with moderate asthma and to assess the effects of corticosteroids and phosphatidylinositol 3-kinase (PI3K) inhibitors.

RESULTS

Sputum macrophages from asthmatic patients expressed significantly more CCL17 mRNA but less CD163 than macrophages from healthy subjects. However, none of the other M2 biomarkers were differentially expressed in asthmatic patients, and ex vivo BAL cells spontaneously produced similar amounts of M2 cytokines/chemokines (IL-10, CCL17, and CCL22). CCL17 mRNA overexpression correlated weakly but significantly with sputum eosinophilia (P = .0252) and was also observed in macrophages from patients with moderate asthma treated with inhaled steroids, suggesting relative insensitivity to inhibition by corticosteroids. The PI3K inhibitor LY294002 inhibited basal CCL17 release from BAL cells and IL-4-stimulated release from MDMs.

CONCLUSIONS

This study does not support the existence in human asthma of the full M2 phenotype described to date but points to upregulation of CCL17 in both patients with mild and those with moderate asthma, providing a further source for this ligand of CCR4(+) cells that contributes to airways inflammation. CCL17 expression is corticosteroid resistant but suppressed by PI3K enzyme inhibitors.

The promiscuous D6 receptor binds several inflammatory CC chemokines and has been recently proposed to act as a chemokine-scavenging decoy receptor. The present study was designed to better characterize the spectrum of CC chemokines scavenged by D6, focusing in particular on CCR4 ligands and analyzing the influence of NH(2)-terminal processing on recognition by this promiscuous receptor. Using D6 transfectants, it was found that D6 efficiently bound and scavenged most inflammatory CC chemokines (CCR1 through CCR5 agonists). Homeostatic CC chemokines (CCR6 and CCR7 agonists) were not recognized by D6. The CCR4 agonists CC chemokine ligand 17 (CCL17) and CCL22 bound to D6 with high affinity. CCL17 and CCL22 have no agonistic activity for D6 (chemotaxis and calcium fluxes), but were rapidly scavenged, resulting in reduced chemotactic activity on CCR4 transfectants. CD26 mediates NH(2) terminus processing of CCL22, leading to the production of CCL22 (3-69) and CCL22 (5-69) that do not interact with CCR4. These NH(2)-terminal truncated forms of CCL22 were not recognized by D6. The results presented in this study show that D6 recognizes and scavenges a wide spectrum of inflammatory CC chemokines, including the CCR4 agonists CCL22 and CCL17. However, this promiscuous receptor is not engaged by CD26-processed, inactive, CCL22 variants. By recognizing intact CCL22, but not its truncated variants, D6 expressed on lymphatic endothelial cells may regulate the traffic of CCR4-expressing cells, such as dendritic cells.