Lee Sullivan

CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue.

Chemokine-directed migration of leukocyte subsets may contribute to the qualitative differences between systemic and mucosal immunity. Here, we demonstrate that in mice lacking the chemokine receptor CCR6, dendritic cells expressing CD11c and CD11b are absent from the subepithelial dome of Peyers patches. These mice also have an impaired humoral immune response to orally administered antigen and to the enteropathic virus rotavirus. In addition, CCR6(-/-) mice have a 2-fold to 15-fold increase in cells of select T lymphocyte populations within the mucosa, including CD4+ and CD8+ alphabeta-TCR T cells. By contrast, systemic immune responses to subcutaneous antigens in CCR6(-/-) mice are normal. These findings demonstrate that CCR6 is a mucosa-specific regulator of humoral immunity and lymphocyte homeostasis in the intestinal mucosa.

Ubiquitous Transgenic Expression of the IL-23 Subunit p19 Induces Multiorgan Inflammation, Runting, Infertility, and Premature Death

p19, a molecule structurally related to IL-6, G-CSF, and the p35 subunit of IL-12, is a subunit of the recently discovered cytokine IL-23. Here we show that expression of p19 in multiple tissues of transgenic mice induced a striking phenotype characterized by runting, systemic inflammation, infertility, and death before 3 mo of age. Founder animals had infiltrates of lymphocytes and macrophages in skin, lung, liver, pancreas, and the digestive tract and were anemic. The serum concentrations of the proinflammatory cytokines TNF-α and IL-1 were elevated, and the number of circulating neutrophils was increased. In addition, ubiquitous expression of p19 resulted in constitutive expression of acute phase proteins in the liver. Surprisingly, liver-specific expression of p19 failed to reproduce any of these abnormalities, suggesting specific requirements for production of biologically active p19. Bone marrow transfer experiments showed that expression of p19 by hemopoietic cells alone recapitulated the phenotype induced by its widespread expression, pointing to hemopoietic cells as the source of biologically active p19. These findings indicate that p19 shares biological properties with IL-6, IL-12, and G-CSF and that cell-specific expression is required for its biological activity.

Generation and analysis of mice lacking the chemokine fractalkine.

ABSTRACT Fractalkine (CX3CL1) is the first described chemokine that can exist either as a soluble protein or as a membrane-bound molecule. Both forms of fractalkine can mediate adhesion of cells expressing its receptor, CX3CR1. This activity, together with its expression on endothelial cells, suggests that fractalkine might mediate adhesion of leukocytes to the endothelium during inflammation. Fractalkine is also highly expressed in neurons, and its receptor, CX3CR1, is expressed on glial cells. To determine the biologic role of fractalkine, we used targeted gene disruption to generate fractalkine-deficient mice. These mice did not exhibit overt behavioral abnormalities, and histologic analysis of their brains did not reveal any gross changes compared to wild-type mice. In addition, these mice had normal hematologic profiles except for a decrease in the number of blood leukocytes expressing the cell surface marker F4/80. The cellular composition of their lymph nodes did not differ significantly from that of wild-type mice. Similarly, the responses offractalkine−/− mice to a variety of inflammatory stimuli were indistinguishable from those of wild-type mice.

Murine CXCR1 Is a Functional Receptor for GCP-2/CXCL6 and Interleukin-8/CXCL8

Functional interleuin-8 (IL-8) receptors (IL-8RA and IL-8RB: CXCR1 and CXCR2, respectively) have been described in human, monkey, dog, rabbit, and guinea pig. Although three IL-8R homologues have been found in rat, only one of these, rat CXCR2, appears to be functional based on responsiveness to ligands. Similarly, CXC chemokines induce biological responses through the murine homolog of CXCR2, but the identification of functional rodent CXCR1 homologues has remained elusive. We have identified and characterized the mouse CXCR1 homologue (mCXCR1). Murine CXCR1 shares 68 and 88% amino acid identity with its human and rat counterparts, respectively. Similar to the tissue distribution pattern of rat CXCR1, we found murine CXCR1 mRNA expression predominantly in lung, stomach, bone marrow, and leukocyte-rich tissues. In contrast to previous reports, we determined that mCXCR1 is a functional receptor. We show predominant engagement of this receptor by mouse GCP-2/CXCL6, human GCP-2, and IL-8/CXCL8 by binding, stimulation of GTPγS exchange, and chemotaxis of mCXCR1-transfected cells. Furthermore, murine CXCR1 is not responsive to the human CXCR2 ligands ENA-78/CXCL5, NAP-2/CXCL7, GRO-α, -β, -γ/CXCL1–3, or rat CINC-1–3. In addition, we show concomitant elevation of mCXCR1 and its proposed major ligand, GCP-2, positively correlated with paw swelling in murine collagen-induced arthritis. This report represents the first description of a functional CXCR1-like receptor in rodents.

The effect of GM-CSF and G-CSF on human neutrophil function

A direct comparison of GM-CSF and G-CSF in a panel of in vitro neutrophil-function assays was performed to investigate any differences in activity profiles. In our modified chemotactic assay, GM-CSF rapidly increased the migratory capacity of polymorphonuclear cells (PMNs) to move toward fMLP and LTB4. In contrast, G-CSF only stimulated PMN migration towards fMLP. GM-CSF, but not G-CSF, increased PMN cytotoxic killing of C. albicans blastospores. The expression of PMN surface antigens associated with Fc- and complement-mediated cell-binding (Fc gamma R1, CR-1 and CR-3), and adhesion signalling (ICAM-1), was increased after the exposure of GM-CSF, but not to G-CSF. In contrast these CSFs demonstrated relative equipotency in their ability to induce PMN anti-bacterial phagocytosis, and to restore the Staphylococcus aureus killing capacity of dexamethasone-suppressed neutrophils. The phagocytic activity of PMNs for opsonized yeast, as well as hexose-monophosphate shunt activity, was equivalent following GM-CSF or G-CSF treatment. We discuss the significance of the difference in activity profiles in this article.

Central Nervous System Inflammation and Neurological Disease in Transgenic Mice Expressing the CC Chemokine CCL21 in Oligodendrocytes

To study the biological role of the chemokine ligands CCL19 and CCL21, we generated transgenic mice expressing either gene in oligodendrocytes of the CNS. While all transgenic mice expressing CCL19 in the CNS developed normally, most (18 of 26) of the CCL21 founder mice developed a neurological disease that was characterized by loss of landing reflex, tremor, and ataxia. These neurological signs were observed as early as postnatal day 9 and were associated with weight loss and death during the first 4 wk of life. Microscopic examination of the brain and spinal cord of CCL21 transgenic mice revealed scattered leukocytic infiltrates that consisted primarily of neutrophils and eosinophils. Additional findings included hypomyelination, spongiform myelinopathy with evidence of myelin breakdown, and reactive gliosis. Thus, ectopic expression of the CC chemokine CCL21, but not CCL19, induced a significant inflammatory response in the CNS. However, neither chemokine was sufficient to recruit lymphocytes into the CNS. These observations are in striking contrast to the reported activities of these molecules in vitro and may indicate specific requirements for their biological activity in vivo.

Increased responsiveness of murine eosinophils to MIP-1β (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‐1α (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‐1β (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‐1α (CCL3) and eotaxin (CCL11) but not MCP‐1 (CCL2), MDC (CCL22), MIP‐1β (CCL4), and TCA‐3 (CCL1). Antigen‐elicited eosinophils migrated toward MIP‐1α (CCL3) and eotaxin (CCL11), but also migrated in response to MIP‐1β (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‐α 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‐1β 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‐1β (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.

IL-4 induces neutrophilic maturation of HL-60 cells and activation of human peripheral blood neutrophils

IL‐4 is a T‐helper cell derived cytokine that has effects on myelomonocytic cell maturation and activation. We have studied the effect of IL‐4 on neutrophilic maturation using the cell line HL‐60 and found that it has a profound effect on the maturation and activation of the cell line. The treatment of HL‐60 cells with recombinant hu IL‐4 (0.15 to 15.0 ng/ml) induced a shift in the percentage of HL‐60 cells staining positive for chloroacetate esterase enzyme activity (indicating commitment to the neutrophilic lineage). IL‐4 increased surface expression of the neutrophil‐lineage antigen WEM G11, the complement receptors CR3 (CD11b) and CR1 (CD35), but not for the monocyte differentiation antigen CD14. IL‐4 treated HL‐60 cells demonstrated enhanced Fc‐ and complement‐mediated phagocytic capacity and increased hexose‐monophosphate shunt activity. In addition, IL‐4 was capable of sustaining the neutrophil maturation of HL‐60 cells that had been pre‐treated for 24h with DMSO. To investigate the effect of IL‐4 on the mature neutrophil, we studied freshly isolated and rested human peripheral blood neutrophils. In the absence of other stimuli, neutrophils were induced by IL‐4 to have significantly elevated phagocytic responses. The response was specific since treatment with anti‐human IL‐4 abolished phagocytic stimulation. Finally, IL‐4 treatment also stimulated resting neutrophils to migrate toward zymosan‐activated serum (ZAS) and human IL‐5. The results demonstrate that IL‐4 is a potent maturation factor for myelocytes to become neutrophils and that IL‐4 can stimulate resting mature neutrophils.

Leukocytes Expressing Green Fluorescent Protein as Novel Reagents for Adoptive Cell Transfer and Bone Marrow Transplantation Studies

Transgenic mice expressing green fluorescent protein (GFP) were generated to provide a source of labeled leukocytes for cell transfer studies. The transgene comprises the GFP coding region under the transcriptional control of the chicken ss-actin promoter and human cytomegalovirus enhancer. Mice expressing this GFP transgene were generated in the B6D2 and in the 129SvEv backgrounds. Flow cytometric analysis of cells from the blood, spleen, and bone marrow of these transgenic mice revealed that most leukocytes, including dendritic cells and memory T cells, express GFP. In allogeneic cell transfers, donor GFP+ splenocytes were detected in the spleen and mesenteric lymph nodes of recipient mice within 2 hours after transfer and for at least 9 days thereafter. In syngeneic experiments using 129-derived GFP+ donor splenocytes, donor cells were detected in multiple tissues of 129 recipients from 2 hours to 3 weeks after transfer. In bone-marrow transplantation experiments using irradiated allogeneic recipients, the percent of GFP+ donor cells in recipients at 3 weeks was comparable to that seen in similar tissues of GFP+ donor mice. These data demonstrate that GFP+ transgenic mice provide a ready source of GFP-expressing primary cells that can be easily monitored after their transfer to recipient animals.

The effects of colony stimulating factors on human monocyte cell function.

We used a panel of functional assays to compare directly the pattern and potency of GM-CSF and M-CSF on monocyte activity associated with cell-mediated immune defense. GM-CSF and M-CSF were found to be equivalent both in their capacity to stimulate human monocyte functions in vitro and in their pattern of monocyte activation. The two CSFs were effective in inducing monocyte chemotaxis towards either fMLP or LTB4 at equivalent concentrations across a panel of donors. GM-CSF and M-CSF demonstrated equipotency in the induction of monocyte phagocytosis of heat-killed bakers yeast and in the regulation of the hexose-monophosphate shunt (NBT reduction). Both were also found to be equivalent in preventing steroid (dexamethasone)-induced suppression of monocyte anti-bacterial (Candida albicans) and anti-fungal (Staphylococcus aureus) phagocytic capacities. GM-CSF was somewhat more effective than M-CSF in stimulating monocyte C. albicans killing at a lower E:T ratio.