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Chemokines are thought to control lymphocyte recruitment to the inflamed endothelium. To dissect chemokine-mediated adhesion, binding of ex vivo isolated splenocytes to tumor necrosis factor (TNF)–activated endothelial cells was analyzed under shear stress. We observed specific adhesion of naive follicular B cells, which could be blocked by pertussis toxin. This indicated a G protein–mediated binding and pointed at a contribution of chemokine receptors to B-cell adhesion. Analysis of chemokines expressed by TNF-activated endothelial cells showed that CC chemokine ligand 2 (CCL2), CCL17, and CCL20 were up-regulated. Only on follicular B cells was the cognate receptor for CCL20, CC chemokine receptor 6 (CCR6), expressed strongly, and a functional transmigration assay with CCR6-negative B cells demonstrated conclusively the sole signaling of CCL20 through CCR6. Desensitization of CCR6 on naive B cells with CCL20 resulted in receptor down-regulation and reduced B-cell adhesion. We conclude that CCL20 plays a vital role in B-cell adhesion to the inflamed endothelium.

The atypical chemokine receptor CCX-CKR regulates bioavailability of CCL19, CCL21, and CCL25, homeostatic chemokines that play crucial roles in thymic lymphopoiesis. Deletion of CCX-CKR results in accelerated experimental autoimmunity induced by immunization. Here we show that CCX-CKR deletion also increases incidence of a spontaneous Sjögren's syndrome-like pathology, characterized by lymphocytic infiltrates in salivary glands and liver of CCX-CKR−/− mice, suggestive of a defect in self-tolerance when CCX-CKR is deleted. This prompted detailed examination of the thymus in CCX-CKR−/− mice. Negatively selected mature SP cells were less abundant in CCX-CKR−/− thymi, yet expansion of both DP and immature SP cells was apparent. Deletion of CCX-CKR also profoundly reduced proportions of DN3 thymocyte precursors and caused DN2 cells to accumulate within the medulla. These effects are likely driven by alterations in thymic stroma as CCX-CKR−/− mice have fewer cTECs per thymocyte, and cTECs express the highest level of CCX-CKR in the thymus. A profound decrease in CCL25 within the thymic cortex was observed in CCX-CKR−/− thymi, likely accounting for their defects in thymocyte distribution and frequency. These findings identify a novel role for CCX-CKR in regulating cTEC biology, which promotes optimal thymocyte development and selection important for self-tolerant adaptive immunity.

Our previous in vitro studies led to proposals that the atypical chemokine receptor CCX-CKR is a scavenger of CCR7 ligand homeostatic chemokines. In the present study, we generated CCX-CKR−/− mice and confirm this scavenger function in vivo. Compared with wild-type mice, CCX-CKR−/− have a 5-fold increase in the level of CCL21 protein in blood, and 2- to 3-fold increases in CCL19 and CCL21 in peripheral lymph nodes. The effect of these protein increases on immunity was investigated after immunization with MOG35-55 peptide emulsified in complete Freund adjuvant (CFA). The subsequent characteristic paralysis develops with enhanced kinetics and severity in CCX-CKR−/− versus wild-type mice. Despite this effect, antigen-specific immune responses in the draining lymph nodes are diminished in CCX-CKR−/− mice. Instead, the earlier onset of disease is associated with enhanced T-cell priming in the CCX-CKR−/− spleen and a skewing of CD4+ T-cell responses toward Th17 rather than Th1. This observation correlates with increased expression of IL-23 in the CCX-CKR−/− spleen and increased CCL21 levels in the central nervous system postimmunization. The early onset of disease in CCX-CKR−/− mice is reversed by systemic administration of neutralizing anti-CCL21 antibodies. Thus, by regulating homeostatic chemokine bioavailability, CCX-CKR influences the localization, kinetics, and nature of adaptive immune responses in vivo.

TNF is a key cytokine in the effector phase of both graft-versus-host disease (GVHD) and the graft-versus leukemia (GVL) effect after bone marrow transplantation (BMT). TNF neutralizing antibodies are now established as effective therapeutic adjuncts for the treatment of severe acute GVHD. TNF signals through the p55 and p75 TNF receptors (TNFR), which are also receptors for the soluble lymphotoxin homotrimer (LTα3). The membrane-bound lymphotoxin heterotrimeric molecule (LTα1β2) signals through the LTβ receptor. The function of these molecules in GVHD remains unknown. Pharmacological agents are available which block either TNF alone, or both TNF and LTα3 and elucidating the roles of these molecules in GVHD is essential for the design of rational therapeutic strategies. We have employed multiple well established preclinical models of GVHD to investigate the roles of these molecules. We first used a functional bioassay to confirm that a human TNFR:Fc construct was capable of preventing physiological activity of both recombinant murine TNF and LTα3. Irradiated B6D2F1 recipients were then transplanted with allogeneic B6.TNF−/− bone marrow and T cells, then treated with control Ig or the TNFR:Fc. Suprisingly, the TNFR:Fc provided significant protection from GVHD mortality in the absence of donor derived TNF (median survival 25.5 vs 35 days, P 3 logs more LTα than LTβ). We subsequently developed the first reported ELISA to assess murine LTα3 protein and were able to clearly demonstrate LTα3 production by wild-type (WT) CD4 T cells seven days after BMT. To further examine this molecule, we performed experiments using WT, LTα−/− or LTβ−/− donor grafts in two BMT systems (B6 → B6D2F1 and B6 → BALB/c). Recipients of LTα−/− grafts were protected from GVHD (P