The major leukocyte chemotactic and activating factors in the mouse gut lumen are not N-formylpeptide receptor 1 agonists.

Cultured bacteria release N-formylpeptides, which are potent chemoattractants for phagocytic leukocytes acting at G-protein-coupled receptors FPR1 and FPR2. However, the distribution and immunologic activity of these molecules at mucosal surfaces, where large numbers of bacteria are separated from the immune system by epithelium, remain undefined. To investigate this for the gut, we tested leukocyte responses to cell-free gut luminal contents from C57Bl/6 mice fed a chow diet. Small and large intestine contents were able to compete with labeled N-formylpeptide for binding to FPR1, indicating the presence of FPR1 ligands in the gut lumen. Material from both small and large intestine induced robust calcium flux responses by primary FPR1(+) leukocytes (mouse bone marrow cells and splenocytes and human peripheral blood neutrophils and mononuclear cells), as well as chemotactic responses by both mouse bone marrow cells and human peripheral blood neutrophils. However, unlike defined N-formylpeptides, calcium flux responses induced by gut luminal contents were insensitive both to pertussis toxin treatment of leukocytes and to proteinase K digestion of the samples. Moreover, the gut samples were fully active on neutrophils from mice lacking Fpr1, and the kinetics of the calcium flux response differed markedly for neutrophils and peripheral blood mononuclear cells. The active factor(s) could be dialyzed using a 3.5-kDa pore size membrane. Thus, mouse intestinal lumen contains small, potent and highly efficacious leukocyte chemotactic and activating factors that may be distinct from neutrophils and peripheral blood mononuclear cells and distinct from Fpr1 agonists.

Characterization of Fpr-rs8, an atypical member of the mouse formyl peptide receptor gene family.

The formyl peptide receptor gene family encodes G protein-coupled receptors for phagocyte chemoattractants, including bacteria- and mitochondria-derived N-formylpeptides. The human family has 3 functional genes, whereas the mouse family has 7 functional genes and 2 possible pseudogenes (ΨFpr-rs2 and ΨFpr-rs3). Here we characterize ΨFpr-rs2, a duplication of Fpr-rs2. Compared to Fpr-rs2, the ΨFpr-rs2 ORF is 186 nucleotides shorter but 98% identical. Due to a deletion and frame shift, the sequences lack homology from amino acid 219-289. Both transcripts were detected constitutively in multiple immune organs; however, ΨFpr-rs2 was consistently less abundant than Fpr-rs2. LPS induced expression of ΨFpr-rs2, but not Fpr-rs2, in spleen and bone marrow. Both transcripts were detected constitutively in thioglycollate-elicited peritoneal neutrophils, whereas only Fpr-rs2 was detected in thioglycollate-elicited peritoneal macrophages. Both transcripts were induced in LPS-stimulated macrophages. ΨFpr-rs2-GFP fusion protein appeared in cytoplasm but not plasma membrane of transfected HEK 293 cells, whereas Fpr-rs2-GFP labeled only plasma membrane. Survival of ΨFpr-rs2(-/-) mice was 33% shorter than that of wild-type and heterozygous littermates (p < 0.05), but no signature pathology was identified. Since ΨFpr-rs2 is expressed in phagocytes and regulated by bacterial products, and may affect longevity, we propose renaming it Fpr-rs8, an atypical member of the formyl peptide receptor gene family.

CXCL12 signaling is independent of Jak2 and Jak3.

Janus kinases (Jaks) are a small family of cytoplasmic tyrosine kinases, critical for signaling by Type I and II cytokine receptors. The importance of Jaks in signaling by these receptors has been firmly established by analysis of mutant cell lines, the generation of Jak knock-out mice, and the identification of patients with Jak3 mutations. While a number of other ligands that do not bind Type I and II cytokine receptors have also been reported to activate Jaks, the requirement for Jaks in signaling by these receptors is less clear. Chemokines for example, which bind seven transmembrane receptors, have been reported to activate Jaks, and principally through the use of pharmacological inhibitors, it has been argued that Jaks are essential for chemokine signaling. In the present study, we focused on CXCR4, which binds the chemokine CXCL12 or stromal cell-derived factor-1, a chemokine that has been reported to activate Jak2 and Jak3. We found that the lack of Jak3 had no effect on CXCL12 signaling or chemotaxis nor did overexpression of wild-type versions of the kinase. Similarly, overexpression of wild-type or catalytically inactive Jak2 or "knocking-down" Jak2 expression using siRNA also had no effect. We also found that in primary lymphocytes, CXCL12 did not induce appreciable phosphorylation of any of the Jaks compared with cytokines for which these kinases are required. Additionally, little or no Stat (signal transducer and activator of transcription) phosphorylation was detected. Thus, we conclude that in contrast to previous reports, Jaks, especially Jak3, are unlikely to play an essential role in chemokine signaling.

Redirecting migration of T cells to chemokine secreted from tumors by genetic modification with CXCR2.

T-cell-based immunotherapies provide a promising means of cancer treatment although durable antitumor responses are infrequent. A potential reason for these shortcomings may lie in the observed lack of trafficking of specific T cells to tumor. Our increasing knowledge of the process of trafficking involving adhesion molecules and chemokines affords us the opportunity to intervene and correct deficiencies in this process. Chemokines can be expressed by a range of tumors and may serve as suitable targets for directing specific T cells toward tumor. We initially sought to identify which chemokines were produced by a range of human tumor cell lines, and which chemokines and chemokine receptors were expressed by cultured T cells. We identified two chemokines: Growth-Regulated Oncogene-alpha (Gro-alpha; CXCL1) and Regulated on Activation Normal T Cell-Expressed and Secreted (RANTES; CCL5), to be secreted by several human tumor cell lines. Expression was also detected in fine-needle aspirates of melanoma from patients. In addition, we determined the expression of several chemokine receptors on cultured human T cells including CCR1, CCR2, CCR4, CCR5, CXCR3, and CXCR4. Cultured, activated human T cells expressed the chemokines lymphotactin (XCL1), RANTES, macrophage inflammatory protein-1 alpha (MIP-1 alpha; CCL3) and MIP-1 beta (CCL4), but no appreciable Gro-alpha. In a strategy to direct T cells toward chemokines expressed by tumors we chose Gro-alpha as the target chemokine because it was produced by tumor and not by T cells themselves. However, T cells did not express the receptor for Gro-alpha, CXCR2, and therefore, T cells were transduced with a retroviral vector encoding CXCR2. Calcium ion mobilization, an important first step in chemokine receptor signaling, was subsequently demonstrated in transduced T cells in response to Gro-alpha. In addition, Gro-alpha was chemotactic for T cells expressing CXCR2 in vitro toward both recombinant protein and tumor-derived chemokine. Interestingly we demonstrate, for the first time, that Gro-alpha was able to induce interferon-gamma (IFN-gamma) secretion from transduced T cells, thereby extending our knowledge of other potential functions of CXCR2. This study demonstrates the feasibility of redirecting the migration properties of T cells toward chemokines secreted by tumors.