David J. Kelvin

Regulation of tyrosine kinase activation and granule release through β-arrestin by CXCR1

Chemoattractant-stimulated granule release from neutrophils, basophils and eosinophils is critical for the innate immune response against infectious bacteria. Interleukin 8 (IL-8) activation of the chemokine receptor CXCR1 was found to stimulate rapid formation of β-arrestin complexes with Hck or c-Fgr. Formation of β-arrestin–Hck complexes led to Hck activation and trafficking of the complexes to granule-rich regions. Granulocytes expressing a dominant-negative β-arrestin–mutant did not release granules or activate tyrosine kinases after IL-8 stimulation. Thus, β-arrestins regulate chemokine-induced granule exocytosis, indicating a broader role for β-arrestins in the regulation of cellular functions than was previously suspected.

beta-arrestins regulate interleukin-8-induced CXCR1 internalization.

The functional role of neutrophils during acute inflammatory responses is regulated by two high affinity interleukin-8 receptors (CXCR1 and CXCR2) that are rapidly desensitized and internalized upon binding their cognate chemokine ligands. The efficient re-expression of CXCR1 on the surface of neutrophils following agonist-induced internalization suggests that CXCR1 surface receptor turnover may involve regulatory pathways and intracellular factors similar to those regulating β2-adrenergic receptor internalization and re-expression. To examine the internalization pathway utilized by ligand-activated CXCR1, a CXCR1-GFP construct was transiently expressed in two different cell lines, HEK 293 and RBL-2H3 cells. While interleukin-8 stimulation promoted CXCR1 sequestration in RBL-2H3 cells, receptor internalization in HEK 293 cells required co-expression of G protein-coupled receptor kinase 2 and β-arrestin proteins. The importance of β-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative β-arrestin 1-V53D mutant to block internalization of CXCR1 in RBL-2H3 cells. A role for dynamin was also demonstrated by the lack of CXCR1 internalization in dynamin I-K44A dominant negative mutant-transfected RBL-2H3 cells. Agonist-promoted co-localization of transferrin and CXCR1-GFP in endosomes of RBL-2H3 cells confirmed that receptor internalization occurs via clathrin-coated vesicles. Our data provides a direct link between agonist-induced internalization of CXCR1 and a requirement for G protein-coupled receptor kinase 2, β-arrestins, and dynamin during this process.

Spatial-Temporal Patterning of Metabotropic Glutamate Receptor-mediated Inositol 1,4,5-Triphosphate, Calcium, and Protein Kinase C Oscillations PROTEIN KINASE C-DEPENDENT RECEPTOR PHOSPHORYLATION IS NOT REQUIRED

The metabotropic glutamate receptors (mGluR), mGluR1a and mGluR5a, are G protein-coupled receptors that couple via Gq to the hydrolysis of phosphoinositides, the release of Ca2+ from intracellular stores, and the activation of protein kinase C (PKC). We show here that mGluR1/5 activation results in oscillatory G protein coupling to phospholipase C thereby stimulating oscillations in both inositol 1,4,5-triphosphate formation and intracellular Ca2+ concentrations. The mGluR1/5-stimulated Ca2+ oscillations are translated into the synchronized repetitive redistribution of PKCβII between the cytosol and plasma membrane. The frequency at which mGluR1a and mGluR5a subtypes stimulate inositol 1,4,5-triphosphate, Ca2+, and PKCβII oscillations is regulated by the charge of a single amino acid residue localized within their G protein-coupling domains. However, oscillatory mGluR signaling does not involve the repetitive feedback phosphorylation and desensitization of mGluR activity, since mutation of the putative PKC consensus sites within the first and second intracellular loops as well as the carboxyl-terminal tail does not prevent mGluR1a-stimulated PKCβII oscillations. Furthermore, oscillations in Ca2+ continued in the presence of PKC inhibitors, which blocked PKCβII redistribution from the plasma membrane back into the cytosol. We conclude that oscillatory mGluR signaling represents an intrinsic receptor/G protein coupling property that does not involve PKC feedback phosphorylation.

Regulation of CCR2 chemokine receptor mRNA stability.

During inflammatory and immunological responses, leukocytes respond to external stimuli by altering the stability of cytokine and cytokine receptor messages. Change in message stability is an effective mechanism for rapidly regulating steady state levels of mRNA. Cytokine messages containing A‐U‐rich elements located in the 3′ untranslated region (ARE) are the best studied examples of this process. AREs have been shown to act as targeting motifs for degradation of cytokine and transcription factor messages. We have recently observed that the interleukin‐8 (IL‐8) receptor messages, IL‐8RA and B (CXCR1 and CXCR2), also undergo changes in stability in response to the inflammatory stimulator lipopolysaccharide (LPS). To determine whether regulation of message stability is a common mechanism for modulation of chemokine receptor mRNA we explored whether the stability of the CC chemokine receptor message for CCR2 (monocyte chemotactic protein‐1 receptor) is also regulated by LPS. We found that LPS induces a rapid loss of steady state levels of CCR2 message through message degradation. Furthermore, LPS stimulated the decay of Poly(A) CCR2 mRNA faster than total CCR2 RNA, indicating that deadenylation is the first step in LPS‐induced CCR2 RNA degradation. We conclude from these experiments that LPS stimulates the rapid degradation of CCR2 messages through a two‐step process, deadenylation followed by degradation of the message body. In contrast to the results obtained for CCR2 mRNA, macrophage inflammatory protein‐1α messages, which contain an ARE motif, were stabilized by LPS stimulation, indicating that chemokine and chemokine receptor mRNA stability are regulated by different and opposing mechanisms. J. Leukoc. Biol.62: 653–660; 1997.

New Strategies for Chemokine Inhibition and Modulation: You Take the High Road and I’ll Take the Low Road

Chemokines are low molecular weight cytokines that induce extravasation, chemotaxis, and activation of a wide variety of leukocytes. Members of the different chemokine families are defined by the orientation of specific critical cysteine residues, and are designated as C-X-C (e.g. interleukin-8), C-C (e.g. regulated upon activation normally T cell expressed and secreted, RANTES), or C (lymphotactin). All chemokines bind to members of a G-protein coupled serpentine receptor superfamily that span the leukocyte cell surface membrane seven times and mediate the biological activities of the individual ligands. Most chemokines possess two major binding surfaces: a high affinity site responsible for specific ligand/receptor interactions and a lower affinity site, also called the heparin-binding or glycosaminoglycan-binding domain, believed to be responsible for the establishment and presentation of chemokine gradients on the surface of endothelial cells and within the extracellular matrix. Although chemokines are clearly beneficial in wound healing, hemopoiesis, and the clearance of infectious organisms, the continued expression of chemokines is associated with chronic inflammation. Therefore, this class of cytokines are attractive targets for the creation of antagonists that abrogate one or more chemokine functions. It is envisioned that such antagonists could serve as a new class of anti-inflammatory drugs. In this commentary, we will discuss two different but related strategies for antagonizing chemokine-induced functions, namely, disruption of the low and high affinity binding sites.

Identification of a novel mechanism for endotoxin-mediated down-modulation of CC chemokine receptor expression.

In the present study, we explored the molecular mechanisms by which bacterial endotoxin (LPS) mediates the down‐regulation of CCR2 receptors on human monocytes. We found that LPS induced a marked reduction in CCR2 cell surface protein levels which was blocked by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A. The effector mechanism underlying LPS‐induced CCR2 down‐modulation appears to involve the enzymatic activity of proteinases since Western blot analysis of LPS‐stimulated monocytes revealed the degradation of a 38‐kDa species corresponding to the CCR2B monomer. In RBL cells expressing the CCR2B‐green fluorescent protein (GFP) fusion chemokine receptor, LPS stimulated the internalization and degradation of CCR2. The serine proteinase inhibitor N‐α‐p‐tosyl‐L‐lysine chloromethyl ketone blocked LPS‐induced down‐modulation of CCR2 in monocytes and CCR2B‐GFP in RBL cells. This work describes a previously uncharacterized mechanism for CC chemokine receptor down‐modulation that is dependent upon tyrosine kinase activation and serine proteinase‐mediated receptor degradation and may provide further insight into the mechanisms of leukocyte regulation during immunological and inflammatory responses.

Genomic Organization and Evolution of the CX3CR1/CCR8 Chemokine Receptor Locus

The chemokine receptors CCR8 and CX3CR1 are key players in adaptive immunity and are co-receptors for human immunodeficiency virus. We describe here the genomic organization and evolutionary history of both of these genes. CX3CR1 has three promoters that transcribe three separate exons that are spliced with a fourth exon containing the coding region. CCR8 has two promoters. One promoter produces a transcript of two spliced exons, and the other promoter transcribes an exon containing the coding region and lacks introns. We analyzed these promoters in the context of a luciferase reporter and identified several positive and negative regulatory elements. Identification of the genomic organization of these genes in mouse demonstrates a similar organization forCCR8, but mouse CX3CR1 lacks two of the human promoters and has an additional mouse-specific promoter that transcribes only the exon containing the coding region and therefore resembles the organization of the human and mouse CCR8genes. We also identify two nontranscribed regions that are highly conserved between human and mouse CX3CR1 containing possible regulatory elements. Examination of the CX3CR1 andCCR8 genes and surrounding genomic regions indicates that these genes are the result of the duplication of an ancestral gene prior to the divergence of teleost fish. We characterize single nucleotide polymorphisms in the promoters of human CCR8 andCX3CR1 and establish linkage relationships betweenCX3CR1 promoter polymorphisms and two previously describedCX3CR1 coding polymorphisms associated with human immunodeficiency virus disease progression and arteriosclerosis susceptibility.

CD45 modulation of CXCR1 and CXCR2 in human polymorphonuclear leukocytes

All leukocytes express the cell surface glycoprotein CD45, which has intrinsic intracellular protein tyrosine phosphatase activity. CD45 is known to play a regulatory role in activation‐induced signaling in lymphocytes; however, little is known of its role in non‐lymphoid leukocytes. Therefore, we examined the potential effect of CD45 on chemokine‐induced signaling in human neutrophils (polymorphonuclear cells, PMN). Treating isolated PMN for 2 h with an anti‐CD45RB antibody (Bra11) down‐modulated expression of the chemokine receptors CXCR1 and CXCR2 to 44 ± 10 % and 47 + 9 % of their respective controls. The tyrosine kinase inhibitors genistein and herbimycin A significantly inhibited the Bra11‐induced down‐modulation of CXCR1 and CXCR2. Furthermore, Bra11 treated PMN were functionally inhibited in their capacity to exhibit IL‐8‐induced transient intracellular Ca2+ increases. Selected targeting of CXC receptors is indicated by the fact that N‐formyl‐Met‐Leu‐Phe (fMLP) receptor expression and function were not lost following Bra11 treatment. The effect of Bra11 on IL‐8‐mediated function and receptor expression was paralleled by decreased tyrosine phosphorylation of a 54‐ to 60‐kDa protein. These findings indicate that CD45 can act to modulate PMN responses to chemokines; thus agents regulating CD45 can potentially modulate leukocyte traffic and may represent a novel therapeutic approach towards the treatment of inflammatory diseases.

Assignment of genes for interleukin-8 receptors (IL8R) A and B to human chromosome band 2q35.

The human genes encoding the interleukin 8 receptors type A and B were assigned to chromosome 2 by polymerase chain reaction amplification and by Southern analysis of a panel of human x rodent somatic cell hybrid DNAs. The IL8R genes were further localized by in situ hybridization to band 2q35.

Treatment with a short course of LF 15-0195 and continuous cyclosporin A attenuates acute xenograft rejection in a rat-to-mouse cardiac transplantation model.

Abstract: Searching for a novel immunosuppressive agent to effectively prevent acute vascular rejection (AVR) is essential for success in clinical xenotransplantation. We previously reported that Lewis rat hearts transplanted into BALB/c mice developed typical AVR in 6 days. The present study was undertaken to determine the efficacy of LF 15‐0195, a new immunosuppressive analog of 15‐deoxyspergualin in the prevention of AVR in a rat‐to‐mouse cardiac xenograft model. We transplanted 2‐week old Lewis rat hearts into BALB/c mice. Four groups were included in this study: untreated recipients and cyclosporin A (CsA) treated recipients were controls; LF 15‐0195 treated recipients or LF 15‐0195 combined with CsA treated recipients were experimental groups. Mouse recipients received either LF 15‐0195 2 mg/kg subcutaneously from day‐1 to post‐operative day 14, or CsA 15 mg/kg subcutaneously daily, from day 0 to endpoint rejection, or the two drugs in combination. We observed that high dose CsA did not inhibit AVR and the graft was rejected in 11.3 ± 1.9 days. Graft histology and immunohistology showed typical AVR, characterized by interstitial hemorrhage, intravascular fibrin deposition, thrombosis, and massive deposition of anti‐rat immunoglobulin G (IgG) and immunoglobulin M (IgM). Serum xenoreactive antibodies (xAbs) were markedly elevated in these animals as well. In contrast, we observed that treatment with LF 15‐0195 alone significantly prolonged graft survival to 19.3 ± 0.7 days. Notably, xAbs were significantly decreased and the rejection pattern of these grafts was cell‐mediated rejection (CMR), instead of AVR. When CsA was combined with LF 15‐0195, the graft mean survival time was further increased to 58.5 ± 17.3 days. Antibody production and T‐cell infiltration were significantly inhibited at the terminal stages of graft survival and pathology showed striking attenuation of both AVR and CMR. Sequential studies on days 6 and 14 demonstrated that LF 15‐0195 either alone or combined with CsA completely inhibited antibody production. However, intragraft infiltration by Mac‐1 positive cells including natural killer cells, macrophages and granulocytes in LF 15‐0195 treated recipients was similar to that of untreated recipients. We conclude that LF 15‐0195 effectively prevented AVR by markedly inhibiting the production of anti‐donor IgG xAbs. Also, treatment with short course LF 15‐0195 and continuous CsA significantly reduced T‐cell infiltration. Studies to test this therapy in inhibiting AVR in a pig‐to‐non‐human primate xenotransplantation model are underway.