Brent Johnston

Bonzo/CXCR6 expression defines type 1–polarized T-cell subsets with extralymphoid tissue homing potential

Chemokine receptor expression is finely controlled during T-cell development. We show that newly identified chemokine receptor Bonzo/CXCR6 is expressed by subsets of Th1 or T-cytotoxic 1 (Tc1) cells, but not by Th2 or Tc2 cells, establishing Bonzo as a differential marker of polarized type 1 T cells in vitro and in vivo. Priming of naive T cells by dendritic cells induces expression of Bonzo on T cells. IL-12 enhances this dendritic cell-dependent upregulation, while IL-4 inhibits it. In blood, 35-56% of Bonzo+ CD4 T cells are Th1 cells, and 60-65% of Bonzo+ CD8 T cells are Tc1 cells, while few Bonzo+ cells are type 2 T cells. Almost all Bonzo+ Tc1 cells contain preformed granzyme A and display cytotoxic effector phenotype. Most Bonzo+ T cells lack L-selectin and/or CCR7, homing receptors for lymphoid tissues. Instead, Bonzo+ T cells are dramatically enriched among T cells in tissue sites of inflammation, such as rheumatoid joints and inflamed livers. Bonzo may be important in trafficking of effector T cells that mediate type 1 inflammation, making it a potential target for therapeutic modulation of inflammatory diseases.

A Common Mucosal Chemokine (Mucosae-Associated Epithelial Chemokine/CCL28) Selectively Attracts IgA Plasmablasts

IgA immunoblasts can seed both intestinal and nonintestinal mucosal sites following localized mucosal immunization, an observation that has led to the concept of a common mucosal immune system. In this study, we demonstrate that the mucosae-associated epithelial chemokine, MEC (CCL28), which is expressed by epithelia in diverse mucosal tissues, is selectively chemotactic for IgA Ab-secreting cells (ASC): MEC attracts IgA- but not IgG- or IgM-producing ASC from both intestinal and nonintestinal lymphoid and effector tissues, including the intestines, lungs, and lymph nodes draining the bronchopulmonary tree and oral cavity. In contrast, the small intestinal chemokine, TECK (CCL25), attracts an overlapping subpopulation of IgA ASC concentrated in the small intestines and its draining lymphoid tissues. Surprisingly, T cells from mucosal sites fail to respond to MEC. These findings suggest a broad and unifying role for MEC in the physiology of the mucosal IgA immune system.

Chronic inflammation upregulates chemokine receptors and induces neutrophil migration to monocyte chemoattractant protein-1

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that stimulates monocyte recruitment when injected into tissues of healthy animals. However, the function of this chemokine in models with preexisting inflammation is not known. Therefore, MCP-1 was superfused over the mesentery of naive rats or rats with chronic adjuvant-induced vasculitis. MCP-1 elicited increased leukocyte transendothelial migration in adjuvant-immunized rats compared with naive animals. Surprisingly, histology revealed that neutrophils constituted the majority of leukocytes recruited in adjuvant-immunized animals. In vitro, MCP-1 was also able to induce chemotaxis of neutrophils isolated from adjuvant-immunized rats but not from naive rats. Flow cytometry revealed novel expression of the CC chemokine receptors CCR1 and CCR2 on neutrophils from adjuvant-immunized animals. In naive animals, an antibody against CD18 blocked leukocyte adhesion and emigration in response to MCP-1. In adjuvant-immunized animals, leukocyte adhesion was reduced by antibodies against the alpha4-integrin but not by antibodies against CD18. However, the CD18 antibody did block emigration. To our knowledge, this study is the first to show increased sensitivity to a CC chemokine in a model with preexisting inflammation, and altered leukocyte recruitment profiles in response to MCP-1. It also demonstrates that CD18 is required for chemokine-induced leukocyte transendothelial migration, independent of its known role in mediating firm adhesion. J. Clin. Invest. 103:1269-1276 (1999).

Differential chemokine responses and homing patterns of murine TCR alpha beta NKT cell subsets.

NKT cells play important roles in the regulation of diverse immune responses. Therefore, chemokine receptor expression and chemotactic responses of murine TCRαβ NKT cells were examined to define their homing potential. Most NKT cells stained for the chemokine receptor CXCR3, while >90% of Vα14i-positive and ∼50% of Vα14i-negative NKT cells expressed CXCR6 via an enhanced green fluorescent protein reporter construct. CXCR4 expression was higher on Vα14i-negative than Vα14i-positive NKT cells. In spleen only, subsets of Vα14i-positive and -negative NKT cells also expressed CXCR5. NKT cell subsets migrated in response to ligands for the inflammatory chemokine receptors CXCR3 (monokine induced by IFN-γ/CXC ligand (CXCL)9) and CXCR6 (CXCL16), and regulatory chemokine receptors CCR7 (secondary lymphoid-tissue chemokine (SLC)/CC ligand (CCL)21), CXCR4 (stromal cell-derived factor-1/CXCL12), and CXCR5 (B cell-attracting chemokine-1/CXCL13); but not to ligands for other chemokine receptors. Two NKT cell subsets migrated in response to the lymphoid homing chemokine SLC/CCL21: CD4− Vα14i-negative NKT cells that were L-selectinhigh and enriched for expression of Ly49G2 (consistent with the phenotype of most NKT cells found in peripheral lymph nodes); and immature Vα14i-positive cells lacking NK1.1 and L-selectin. Mature NK1.1+ Vα14i-positive NKT cells did not migrate to SLC/CCL21. BCA-1/CXCL13, which mediates homing to B cell zones, elicited migration of Vα14i-positive and -negative NKT cells in the spleen. These cells were primarily CD4+ or CD4−CD8− and were enriched for Ly49C/I, but not Ly49G2. Low levels of chemotaxis to CXCL16 were only detected in Vα14i-positive NKT cell subsets. Our results identify subsets of NKT cells with distinct homing and localization patterns, suggesting that these populations play specialized roles in immunological processes in vivo.

Role for CXCR6 in Recruitment of Activated CD8+ Lymphocytes to Inflamed Liver

Hepatic infiltration of activated CD8 lymphocytes is a major feature of graft-vs-host disease (GvHD). Chemoattractant cytokines and their receptors are key regulators of lymphocyte trafficking, but the involvement of chemoattractant receptors in the physiologic recruitment of cells into the inflamed liver has not been defined. The present study examines the role of the chemokine receptor CXCR6, which is highly expressed by liver-infiltrating CD8 T cells. Hepatic accumulation of donor CD8, but not donor CD4, lymphocytes was significantly reduced in GvHD induced by transfer of CXCR6−/−, H-2Db lymphocytes into BDF1, H-2Dbxd recipients. To determine whether altered recruitment contributes to the reduced accumulation, CXCR6−/− or wild-type splenic lymphocytes participating in an active GvHD response were isolated and transferred i.v. into secondary recipients with active GvHD, and the short term (6-h) recruitment of transferred cells to the inflamed liver was assessed. CXCR6−/− CD8 (but not CD4) cells displayed a significant (33%) reduction in liver localization, whereas frequencies in blood of CXCR6−/− and wild-type CD8 cells were similar. Proliferation and apoptosis of liver-infiltrating donor CD8 cells were unaffected. We conclude that CXCR6 helps mediate the recruitment of activated CD8 lymphocytes in GvHD-induced hepatitis and may be a useful target to treat pathological inflammation in the liver.

Distinct subsets of human Vα24-invariant NKT cells: cytokine responses and chemokine receptor expression

CD1d-restricted T-cell receptor Valpha-invariant natural killer T (NKT) cells are important regulators of immune responses through their efficient secretion of T helper 1 (Th1) and Th2 cytokines. Recently, it has been shown that this NKT-cell population contains functionally distinct subsets, producing different sets of cytokines and cytotoxic effector molecules. NKT-cell subsets are also distinct from each other in their expression of adhesion molecules and chemokine receptors, suggesting that they might be targeted to different tissues and perform different immune functions.

Leukotriene C4/D4 Induces P-Selectin and Sialyl Lewisx–Dependent Alterations in Leukocyte Kinetics In Vivo

The objective of this study was to assess the effect of leukotriene C4 (LTC4) on the flux of rolling leukocytes, leukocyte rolling velocity, and leukocyte adhesion in postcapillary venules in vivo and to study the underlying molecular mechanisms involved. LTC4 (20 nmol/L) induced a rapid and significant increase in leukocyte rolling flux that was inhibitable by an anti-P-selectin antibody and soluble sialyl Lewis(x) (sLe(x)). LTC4 also induced a significant reduction in leukocyte rolling velocity, an event that was independent of P-selectin but entirely dependent on sLe(x). This LTC4-induced reduction in leukocyte rolling velocity was independent of any hemodynamic alterations. Another P-selectin effector, histamine, did not affect leukocyte rolling velocity even at > 5000 times the concentration of LTC4. Treatment with an anti-L-selectin antibody had no effect on the LTC4-induced increase in leukocyte rolling or reduction in rolling velocity. Inhibition of LTC4 bioconversion to LTD4 by pretreatment with L-serine (100 mumol/L) prevented the LTC4-induced increase in leukocyte rolling flux and the LTC4-induced reduction in leukocyte rolling velocity. A subtle, yet significant, increase in leukocyte adhesion was also observed with LTC4. Pretreatment with a platelet-activating factor receptor antagonist returned the LTC4-induced leukocyte rolling velocity to baseline levels. The addition of a very low concentration of platelet-activating factor (1 nmol/L) induced significant leukocyte adhesion in the presence of LTC4 but not histamine.(ABSTRACT TRUNCATED AT 250 WORDS)

A Critical Role for Mast Cells and Mast Cell-Derived IL-6 in TLR2-Mediated Inhibition of Tumor Growth

Several TLR agonists are effective in tumor immunotherapy, but their early innate mechanisms of action, particularly those of TLR2 agonists, are unclear. Mast cells are abundant surrounding solid tumors where they are often protumorigenic and enhance tumor angiogenesis. However, antitumor roles for mast cells have also been documented. The impact of mast cells may be dependent on their activation status and mediator release in different tumors. Using an orthotopic melanoma model in wild-type C57BL/6 and mast cell-deficient KitW-sh/W-sh mice and a complementary Matrigel–tumor model in C57BL/6 mice, mast cells were shown to be crucial for TLR2 agonist (Pam3CSK4)-induced tumor inhibition. Activation of TLR2 on mast cells reversed their well-documented protumorigenic role. Tumor growth inhibition after peritumoral administration of Pam3CSK4 was restored in KitW-sh/W-sh mice by local reconstitution with wild-type, but not TLR2-deficient, mast cells. Mast cells secrete multiple mediators after Pam3CSK4 activation, and in vivo mast cell reconstitution studies also revealed that tumor growth inhibition required mast cell-derived IL-6, but not TNF. Mast cell-mediated anticancer properties were multifaceted. Direct antitumor effects in vitro and decreased angiogenesis and recruitment of NK and T cells in vivo were observed. TLR2-activated mast cells also inhibited the growth of lung cancer cells in vivo. Unlike other immune cells, mast cells are relatively radioresistant making them attractive candidates for combined treatment modalities. This study has important implications for the design of immunotherapeutic strategies and reveals, to our knowledge, a novel mechanism of action for TLR2 agonists in vivo.

Critical Role for the Chemokine Receptor CXCR6 in Homeostasis and Activation of CD1d-Restricted NKT Cells

NK T (NKT) cells play important roles in the regulation of diverse immune responses. However, little is known about the mechanisms that regulate homeostasis and activation of these cells. Thymic NKT cells up-regulated the chemokine receptor CXCR6 following positive selection and migrated toward CXCL16 in vitro. However, CXCR6 was not essential for thymic development or maturation. In contrast, liver and lung NKT cells were depleted in CXCR6+/− and CXCR6−/− mice. The reduction in liver and lung NKT cells coincided with an increase in bone marrow NKT cells, suggesting a redistribution of NKT cells in CXCR6−/− animals. In wild-type mice, CXCL16 neutralization reduced accumulation of mature NK1.1+, but not immature NK1.1− NKT cell recent thymic emigrants in the liver. Given that thymic NKT cells are preferentially exported as NK1.1− cells, this suggests an additional role for CXCR6/CXCL16 in maturation or survival of immature liver NKT cells. CXCL16 blockade did not deplete resident NK1.1+ NKT cells, indicating that CXCR6/CXCL16 are not required to retain mature NKT cells in the liver. Cytokine production by liver and spleen NKT cells was impaired in CXCR6−/− mice following in vivo stimulation with α-galactosylceramide, implicating a novel role for CXCR6 in NKT cell activation. Reduced IFN-γ production was not due to an intrinsic defect as production was normal following PMA and ionomycin stimulation. Preformed transcripts for IL-4, but not IFN-γ, were reduced in CXCR6−/− liver NKT cells. These data identify critical roles for CXCR6/CXCL16 in NKT cell activation and the regulation of NKT cell homeostasis.

The α4-integrin: an alternative pathway for neutrophil recruitment?

Abstract In general, neutrophils are assumed not to express the α 4 -integrin, a molecule implicated in the recruitment of mononuclear leukocytes and eosinophils to sites of inflammation. In this article, the authors review evidence demonstrating that neutrophils can express the α 4 -integrin and use this alternative mechanism to mediate neutrophil adhesion and migration.