Examination of the function of RANTES, MIP-1alpha, and MIP-1beta following interaction with heparin-like glycosaminoglycans

Chemokines are a group of small proteins that have a variety of functions, including the activation and recruitment of immune cells during episodes of inflammation. In common with many cytokines, it has been observed that chemokines have the potential to bind heparin-like glycosaminoglycan molecules, which are normally expressed on proteoglycan components of the cell surface and extracellular matrix. The significance of this interaction for chemokine activity remains a subject of debate. In this study, Chinese hamster ovary cells were transfected separately with the human chemokine receptors CCR1 and CCR5, and these receptors were shown to induce an intracytoplasmic Ca(2+) flux and cellular chemotaxis following stimulation with the natural CC chemokine ligands (MIP-1alpha, RANTES (regulated on activation normal T cell expressed), and MIP-1beta). In further experiments, mutant CHO cells, with a defect in normal glycosaminoglycan (GAG) expression, were also transfected with, and shown to express similar levels of, CCR1 and CCR5. Although these receptors were functional, it was found that the mutant cells required exposure to higher concentrations of ligands than the wild-type cells in order to produce the same intracytoplasmic Ca(2+) flux. Radioligand binding experiments demonstrated that specific chemokine receptors expressed by wild-type cells had a significantly greater affinity for MIP-1alpha than similar receptors expressed by GAG-deficient mutants. However, there was no significant difference between these cells in their affinity for RANTES or MIP-1beta. In conclusion, it has been demonstrated clearly that GAG expression is not necessary for the biological activity of the chemokines MIP-1alpha, RANTES, or MIP-1beta. However, the presence of cell surface GAGs does enhance the activity of low concentrations of these chemokines by a mechanism that appears to involve sequestration onto the cell surface.     

Differential CCR1-mediated chemotaxis signaling induced by human CC chemokine HCC-4/CCL16 in HOS cells

Human CC chemokine-4 (HCC-4)/CCL16 is a chemoattractant for monocytes and lymphocytes. Although HCC-4 binds to multiple CC chemokine receptors, the receptor-mediated signal transduction pathway induced by HCC-4 has not been characterized. Human osteogenic sarcoma cells stably expressing CCR1 were used to investigate HCC-4-mediated chemotaxis signaling events via CCR1. The chemotactic activity of HCC-4 as well as those of other CCR1-dependent chemokines including MIP-1alpha/CCL3, RANTES/CCL5, and Lkn-1/CCL15 was inhibited by the treatment of pertussis toxin, an inhibitor of Gi/Go protein, U73122, an inhibitor of phospholipase C (PLC), and rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta). These results indicate that HCC-4-induced chemotaxis signaling is mediated through Gi/Go protein, PLC, and PKCdelta. SB202190, an inhibitor of p38 mitogen activated protein kinase, only blocked the chemotactic activity of HCC-4, but not those of other CCR1-dependent chemokines. SB202190 inhibited HCC-4-induced chemotaxis in a dose-dependent manner (P < 0.01). HCC-4 induces p38 activation in both a time and dose-dependent manner. However, such p38 activation was not induced by other CCR1-dependent chemokines. To further investigate the differential effect of HCC-4, the Ca2+ mobilization was examined. HCC-4 induced no intracellular Ca2+ flux in contrast to other CCR1-dependent chemokines. These results indicate that HCC-4 transduces signals differently from other CCR1-dependent chemokines and may play different roles in the immune response.     

G protein-coupled chemokine receptors induce both survival and apoptotic signaling pathways

Chemokine receptors are essential for triggering chemotaxis to immune cells; however, a number of them can also mediate death when engaged by nonchemokine ligands. When the chemokine receptor CXCR4 is engaged by stromal cell-derived factor (SDF1)alpha, it triggers cells to chemotax, and in some cell types such as neurons, causes cell death. To elucidate this dual and opposing receptor function, we have investigated whether CXCR4 activation by its chemokine SDF1alpha could lead to the simultaneous activation of both anti- and proapoptotic signaling pathways; the balance ultimately influencing cell survival. CXCR4 activation in CD4 T cells by SDF1alpha led to the activation of the prosurvival second messengers, Akt and extracellular signal-regulated protein kinase. Selective inhibition of each signal demonstrated that extracellular signal-regulated protein kinase is essential for mediating SDF1alpha-triggered chemotaxis but does not confer an antiapoptotic state. In contrast, Akt activation through CXCR4 by SDF1alpha interactions is necessary to confer resistance to apoptosis. The proapoptotic signaling pathway triggered by SDF1alpha-CXCR4 interaction involves the G(ialpha) protein-independent activation of the proapoptotic MAPK (p38). Furthermore, other chemokines and chemokine receptors also signal chemotaxis and proapoptotic effects via similar pathways. Thus, G(ialpha) protein-coupled chemokine receptors can function as death prone receptors and the balance between the above signaling pathways will ultimately mandate the fate of the activated cell.     

Modulation of immunity against herpes simplex virus infection via mucosal genetic transfer of plasmid DNA encoding chemokines

In this study, we examined the effects of murine chemokine DNA, as genetic adjuvants given mucosally, on the systemic and distal mucosal immune responses to plasmid DNA encoding gB of herpes simplex virus (HSV) by using the mouse model. The CC chemokines macrophage inflammatory protein 1beta (MIP-1beta) and monocyte chemotactic protein 1 (MCP-1) biased the immunity to the Th2-type pattern as judged by the ratio of immunoglobulin isotypes and interleukin-4 cytokine levels produced by CD4(+) T cells. The CXC chemokine MIP-2 and the CC chemokine MIP-1alpha, however, mounted immune responses of the Th1-type pattern, and such a response rendered recipients more resistant to HSV vaginal infection. In addition, MIP-1alpha appeared to act via the upregulation of antigen-presenting cell (APC) function and the expression of costimulatory molecules (B7-1 and B7-2), whereas MIP-2 enhanced Th1-type CD4(+) T-cell-mediated adaptive immunity by increasing gamma interferon secretion from activated NK cells. Our results emphasize the value of using the mucosal route to administer DNA modulators such as chemokines that function as adjuvants by regulating the activity of innate immunity. Our findings provide new insight into the value of CXC and CC chemokines, which act on different innate cellular components as the linkage signals between innate and adaptive immunity in mucosal DNA vaccination.     

Biology of chemokine and classical chemoattractant receptors: differential requirements for adhesion-triggering versus chemotactic responses in lymphoid cells

Several chemoattractant receptors can support agonist-induced, integrin- dependent arrest of rolling neutrophils in inflamed venules in vivo, as well as subsequent crawling into tissues. It has been hypothesized that receptors of the Galpha(i)-linked chemoattractant subfamilies, especially receptors for chemokines, may mediate parallel activation- dependent arrest of homing lymphocyte subsets. However, although several chemokines can attract subsets of B or T cells, robust chemoattractant triggering of resting lymphocyte adhesion to vascular ligands has not been observed. To study the biology of individual leukocyte chemoattractant receptors in a defined lymphoid environment, mouse L1/2 pre-B cells and/or human Jurkat T cells were transfected with alpha (IL-8 receptor A) or beta (MIP-1alpha/CC-CKR-1) chemokine receptors, or with the classical chemoattractant C5a (C5aR) or formyl peptide receptors (fPR). All receptors supported robust agonist- dependent alpha4beta1 integrin-mediated adhesion of lymphocytes to VCAM- 1. L1/2 cells cotransfected with fPR and beta7 integrin were also induced to bind MAdCAM-1, suggesting common mechanisms coupling chemoattractant receptors to activation of distinct integrins. Adhesion was rapid but transient, with spontaneous reversion to unstimulated levels within 5 min after peak binding. When observed under flow conditions, alpha4beta1-mediated arrest occurred within seconds after initiation of contact and rolling of IL-8RA transfectants on VCAM-1/IL- 8 co-coated surface; and arrest reversed spontaneously after a mean of 5 min with a return to rolling behavior. Each of the receptors also conferred agonist-specific chemotaxis; however, whereas strong adhesion required simultaneous occupancy of many receptors with maximal responses above the Kd, chemotaxis in each case was suppressed at high agonist concentrations. The findings indicate that alpha and beta chemokine as well as classical chemoattractant receptors can trigger robust adhesion as well as directed migration of lymphoid cells, but that the requirements for and kinetics of adhesion triggering and chemotaxis are distinct, thus permitting their independent regulation. They suggest that the discordance between proadhesive and chemoattractant responses of circulating lymphocytes to many chemokines may reflect quantitative aspects of receptor expression and/or coupling rather than qualitative differences in receptor signaling.     

Proinflammatory chemokines, such as C-C chemokine ligand 3, desensitize mu-opioid receptors on dorsal root ganglia neurons

Pain is one of the hallmarks of inflammation. Opioid receptors mediate antipain responses in both the peripheral nervous system and CNS. In the present study, pretreatment of CCR1: mu-opioid receptor/HEK293 cells with CCL3 (MIP-1alpha) induced internalization of mu-opioid receptors and severely impaired the mu-opioid receptor-mediated inhibition of cAMP accumulation. Immunohistochemical staining showed that CCR1 and mu-opioid receptors were coexpressed on small to medium diameter neurons in rat dorsal root ganglion. Analysis of ligand-induced calcium flux showed that both types of receptors were functional. Pretreatment of neurons with CCL3 exhibited an impaired [D-Ala(2),N-MePhe(4),Gly-o15]enkephalin-elicited calcium response, indicative of the heterologous desensitization of mu-opioid receptors. Other chemokines, such as CCL2, CCL5, and CXCL8, exhibited similar inhibitory effects. Our data indicate that proinflammatory chemokines are capable of desensitizing mu-opioid receptors on peripheral sensory neurons, providing a novel potential mechanism for peripheral inflammation-induced hyperalgesia.     

Chemokines and chemokine receptors in renal transplantation--from bench to bedside

Attraction of mononuclear cells to sites of inflammation requires a close interplay of the inflammatory signal presented via chemokines and specific receptors on effector cells. First studies on acute renal transplant rejection demonstrated the involvement of CC-chemokines, such as RANTES, MIP-1alpha, MIP-1beta and MCP-1, as well as CXC-chemokines such as IL-8 and IP-10, correlating with expression of the corresponding chemokine receptors, CCR1, CCR5 and CCR2 as well as CXCR3. Since then, the pathophysiologic relevance has been extended to chronic allograft nephropathy and transplant glomerulopathy. Chemokine expression can be triggered by different stimuli, e.g. brain death, ischemia, HLA-mismatch and infection. Furthermore, anti-inflammatory chemokines have been identified. Chemokine receptor 7, e.g. enhances homing of lymphocytes to lymphatic tissues and the Duffy antigen receptor, DARC, a non-specific receptor that binds and inactivates different chemokines. While measurement of chemokine expression in clinical transplantation may facilitate the differential diagnosis of allograft dysfunction, knowledge of the chemokine network has also widened the understanding of transplant rejection and opened novel therapeutic approaches. Observations from humans with mutations of the chemokine network as well as transplantation of animals with targeted deletions in this system suggest that manipulations of chemokine signalling may improve the success rates of transplantation. Blocking chemokines unselectively with Met-RANTES or specifically with small molecule inhibitors of various chemokine receptors has lead to improved outcome in animal models. Currently, first human trials are under way to investigate drugs that stimulate lymphocyte homing. Inhibitors of CCR1 and CCR5 are being tested for other human diseases and may eventually be available in transplantation. Nonetheless, chemokine blockade my rather serve as an adjunct in the management of transplant recipients than a new "magic bullet".     

Differential effects of leukotactin-1 and macrophage inflammatory protein-1 alpha on neutrophils mediated by CCR1

The human CC chemokine leukotactin-1 (Lkn-1) is both a strong chemoattractant for neutrophils, monocytes, and lymphocytes and a potent agonist for CCR1 and CCR3. However, human neutrophils do not migrate when the cells are stimulated with other human CC chemokines, such as human macrophage inflammatory protein-1 alpha (hMIP-1 alpha) and eotaxin, which also use the CCR1 and CCR3 as their receptors. In this report, we demonstrate that while hMIP-1 alpha induced a negligible level of calcium flux and chemotaxis, Lkn-1 produced a high level of calcium flux and chemotaxis in human neutrophils. Lkn-1 cross-desensitized hMIP-1 alpha-induced calcium flux, but hMIP-1 alpha had little effect on the Lkn-1-induced response in human neutrophils. The same pattern was observed in peritoneal neutrophils from wild-type mice, whereas neutrophils from CCR1-/- mice failed to respond to either MIP-1 alpha or Lkn-1. Scatchard analysis revealed a single class of receptor for both hMIP-1 alpha and Lkn-1 on human neutrophils with dissociation constants (Kd) of 3.2 nM and 1.1 nM, respectively. We conclude that CCR1 is a receptor mediating responses to both MIP-1 alpha and Lkn-1 on neutrophils and produces different biological responses depending on the ligand bound.     

TGF-beta 1 reciprocally controls chemotaxis of human peripheral blood monocyte-derived dendritic cells via chemokine receptors

We examined the effect of TGF-beta 1 on the chemotactic migratory ability of human monocyte-derived dendritic cells (DCs). Treatment of immature DCs with TGF-beta 1 resulted in increased expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXC chemokine receptor-4 (CXCR-4), which were concomitant with enhanced chemotactic migratory responses to their ligands, RANTES (for CCR-1, CCR-3, and CCR-5), macrophage-inflammatory protein-3 alpha (MIP-3 alpha) (for CCR-6), or stromal cell-derived growth factor-1 alpha (for CXCR-4). Ligation by TNF-alpha resulted in down-modulation of cell surface expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and the chemotaxis for RANTES, MIP-3 alpha, and stromal cell-derived growth factor-1 alpha, whereas this stimulation up-regulated the expression of CCR-7 and the chemotactic ability for MIP-3beta. Stimulation of mature DCs with TGF-beta 1 also enhanced TNF-alpha-induced down-regulation of the expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and chemotaxis to their respective ligands, while this stimulation suppressed TNF-alpha-induced expression of CCR-7 and chemotactic migratory ability to MIP-3 beta. Our findings suggest that TGF-beta 1 reversibly regulates chemotaxis of DCs via regulation of chemokine receptor expression.     

Chemokines and their receptors in the brain: pathophysiological roles in ischemic brain injury

Chemokines constitute a large family of structurally-related small cytokines originally identified as factors regulating the migration of leukocytes in inflammatory and immune responses. Production of chemokines and their receptors in the brain has been reported under various pathological conditions. We revealed that mRNA expression for monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha), members of the CC chemokines, was induced in the rat brain after focal cerebral ischemia, and that intracerebroventricular injection of viral macrophage inflammatory protein-II (vMIP-II), a broad-spectrum chemokine receptor antagonist, reduced infarct volume in a dose-dependent manner. These findings suggest that brain chemokines are involved in ischemic injury, and that chemokine receptors are potential targets for therapeutic intervention in stroke. Another potential target to suppress the harmful effect of chemokines is the signal transmission system(s) regulating the chemokine production. However, very little is known about how the production of chemokines is regulated in the ischemic brain. We examined the induction of MCP-1 production by excitotoxic injury via activation of NMDA receptors in the cortico-striatal slice cultures, and found that excitotoxic injury induced MCP-1 production in the slice culture. Almost all of the MCP-1 immunoreactivity was located on astrocytes. On the other hand, NMDA-treatment failed to increase the MCP-1 production in the enriched astrocyte cultures, indicating that NMDA dose not directly act on astrocytes. Some signal(s) is likely sent from the injured neurons to astrocytes to induce the MCP-1 production. These results showed that organotypic slice cultures are useful to investigate the molecular mechanism regulating the chemokine production in the injured brain.     

Interactions between opioid and chemokine receptors: heterologous desensitization

The opioid and chemokine receptors are both members of the seven transmembrane G protein-coupled receptor (GPCR) superfamily. Desensitization is believed to be a major element of the regulation of the function of these receptors, and recent findings suggest that both agonist-dependent (homologous) desensitization and heterologous desensitization can control receptor activity. The cross-desensitization between opioid and chemokine receptors has significant implications for our understanding of both the regulation of leukocyte trafficking, as well as the regulation of chemokine receptor function in inflammatory disease states. We also review findings which suggest that pro-inflammatory chemokine receptor-induced heterologous desensitization of opioid receptors has important implications for the regulation of opioid receptor function in the nervous system.     

A Model of GAG/MIP-2/CXCR2 Interfaces and Its Functional Effects

MIP-2/CXCL2 is a murine chemokine related to human chemokines that possesses the Glu-Leu-Arg (ELR) activation motif and activates CXCR2 for neutrophil chemotaxis. We determined the structure of MIP-2 to 1.9 ? resolution and created a model with its murine receptor CXCR2 based on the coordinates of human CXCR4. Chemokine-induced migration of cells through specific G-protein coupled receptors is regulated by glycosaminoglycans (GAGs) that oligomerize chemokines. MIP-2 GAG-binding residues were identified that interact with heparin disaccharide I-S by NMR spectroscopy. A model GAG/MIP-2/CXCR2 complex that supports a 2:2 complex between chemokine and receptor was created. Mutants of these disaccharide-binding residues were made and tested for heparin binding, in vitro neutrophil chemotaxis, and in vivo neutrophil recruitment to the mouse peritoneum and lung. The mutants have a 10-fold decrease in neutrophil chemotaxis in vitro. There is no difference in neutrophil recruitment between wild-type MIP-2 and mutants in the peritoneum, but all activity of the mutants is lost in the lung, supporting the concept that GAG regulation of chemokines is tissue-dependent.     

Monocyte chemotactic protein-1 regulates leukocyte recruitment during gastric ulcer recurrence induced by tumor necrosis factor-alpha

TNF-alpha has numerous biological activities, including the induction of chemokine expression, and is involved in many gastric injuries. C-C chemokines [monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha] and C-X-C chemokines [MIP-2 and cytokine-induced neutrophil chemoattractant (CINC)-2alpha] mediate chemotaxis of monocytes and neutrophils, respectively. We examined the roles of TNF-alpha and dynamics of chemokine expression in gastric ulceration including ulcer recurrence and indomethacin-induced injury. Rats with healed chronic gastric ulcers received intraperitoneal TNF-alpha to induce ulcer recurrence. Some rats were given neutralizing antibodies against neutrophils or MCP-1 together with TNF-alpha. In a separate experiment, rats were orally administered 20 mg/kg indomethacin with or without pretreatment with pentoxifylline (an inhibitor of TNF-alpha synthesis) or anti-MCP-1 antibody. TNF-alpha (1 microg/kg) induced gastric ulcer recurrence after 48 h, which was completely prevented by anti-neutrophil antibody. TNF-alpha increased the number of macrophages and MCP-1 mRNA expression in scarred mucosa from 4 h, whereas it increased MPO activities (marker of neutrophil infiltration) and mRNA expression of MIP-2 and CINC-2alpha from 24 h. Anti-MCP-1 antibody inhibited leukocyte infiltration with reduction of the levels of C-X-C chemokines and prevented ulcer recurrence. Indomethacin treatment increased TNF-alpha/chemokine mRNA expression from 30 min and induced macroscopic erosions after 4 h. Pentoxifylline inhibited the indomethacin-induced gastric injury with reduction of neutrophil infiltration and expression of chemokine (MCP-1, MIP-2, and CINC-2alpha). Anti-MCP-1 antibody also inhibited the injury and these inflammatory responses but did not affect TNF-alpha mRNA expression. In conclusion, increased MCP-1 triggered by TNF-alpha may play a key role in gastric ulceration by regulating leukocyte recruitment and chemokine expression.     

Coevolution of the mammalian chemokines and their receptors

 A phylogeny of mammalian chemokines revealed two major clusters, corresponding to the CC and CXC chemokines; the C chemokines appeared to be more closely related to the former. In a phylogeny of chemokine receptors, there were also two major clusters: one containing CC chemokine receptors plus other receptors of unknown function and another containing CXC receptors and other receptors of unknown function. However, within the CC receptors, there was not a close correspondence between the phylogenies of chemokines and their receptors. The CC chemokines contained two major subfamilies: (1) the MIP subfamily (including MIP-1α, MIP-1β, and RANTES); and (2) the MCP subfamily (including MCP-1,-2,-3, and -4 and eotaxin). Receptors having preferred ligands in the MCP subfamily did not constitute a monophyletic group but rather evolved twice independently. Reconstruction of ancestral amino acid sequences suggested that these two groups of MCP receptors did not convergently evolve any amino acid residues; rather, they convergently lost sequence features found in the third and fourth extracellular domains of known receptors for MIP-subfamily chemokines. Received: 1 May 1998 / Revised: 3 July 1998     

Reduced chemokine and chemokine receptor expression in spinal cords of TCR BV8S2 transgenic mice protected against experimental autoimmune encephalomyelitis with BV8S2 protein

The perivascular transmigration and accumulation of macrophages and T lymphocytes in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) may be partly regulated by low m.w. chemotactic cytokines. Using the RNase protection assay and ELISA, we quantified expression of chemokines and chemokine receptors in the spinal cord (SC), brain, and lymph nodes of BV8S2 transgenic mice that developed or were protected from EAE by vaccination with BV8S2 protein. In paralyzed control mice, the SC had increased cellular infiltration and strong expression of the chemokines RANTES, IFN-inducible 10-kDa protein, and monocyte chemoattractant protein-1 and the cognate chemokine receptors CCR1, CCR2, and CCR5, with lower expression of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and MIP-2; whereas brain had less infiltration and a lower expression of a different pattern of chemokines and receptors. In TCR-protected mice, there was a decrease in the number of inflammatory cells in both SC and brain. In SC, the reduced cellular infiltrate afforded by TCR vaccination was commensurate with profoundly reduced expression of chemokines and their cognate chemokine receptors. In brain, however, TCR vaccination did not produce significant changes in chemokine expression but resulted in an increased expression of CCR3 and CCR4 usually associated with Th2 cells. In contrast to CNS, lymph nodes of protected mice had a significant increase in expression of MIP-2 and MIP-1beta but no change in expression of chemokine receptors. These results demonstrate that TCR vaccination results in selective reduction of inflammatory chemokines and chemokine receptors in SC, the target organ most affected during EAE.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit LPS-stimulated MIP-1alpha production and mRNA expression

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides with immunomodulatory properties, including the regulation of several proinflammatory mediators. Such mediators, for example chemokines, influence trafficking of inflammatory cells and contribute to shaping the immune response. In the present work, we studied the effect of VIP and PACAP on the CC chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) production in LPS-stimulated RAW 264.7 macrophage cell line. VIP and PACAP inhibited the production of MIP-1alpha in a dose-dependent manner and over a broad spectrum of LPS concentrations. The use of selective agonists and antagonists of VIP/PACAP receptors showed that type 1 VIP receptor (VPAC1) is the major receptor involved, but the type 2 VIP receptor (VPAC2) may be also implicated. By using selective PKA and PKC inhibitors and cAMP mimicked agents, we demonstrated a cAMP-dependent signalling pathway for the inhibitory effect of VIP/PACAP on MIP-1alpha production, although a minor non-mediated cAMP pathway was also involved. mRNA expression studies showed a down-regulation of MIP-1alpha gene expression by VIP and PACAP. Taken together, the present work strongly supports an anti-inflammatory role of VIP and PACAP by a new mechanism associated with impairment of a key component of the chemokine network.     

LD78beta, a non-allelic variant of human MIP-1alpha (LD78alpha), has enhanced receptor interactions and potent HIV suppressive activity.

Chemokines play diverse roles in inflammatory and non-inflammatory situations via activation of heptahelical G-protein-coupled receptors. Also, many chemokine receptors can act as cofactors for cellular entry of human immunodeficiency virus (HIV) in vitro. CCR5, a receptor for chemokines MIP-1alpha (LD78alpha), MIP-1beta, RANTES, and MCP2, is of particular importance in vivo as polymorphisms in this gene affect HIV infection and rate of progression to AIDS. Moreover, the CCR5 ligands can prevent HIV entry through this receptor and likely contribute to the control of HIV infection. Here we show that a non-allelic isoform of human MIP-1alpha (LD78alpha), termed LD78beta or MIP-1alphaP, has enhanced receptor binding affinities to CCR5 (approximately 6-fold) and the promiscuous beta-chemokine receptor, D6 (approximately 15-20-fold). We demonstrate that a proline residue at position 2 of MIP-1alphaP is responsible for this enhanced activity. Moreover, MIP-1alphaP is by far the most potent natural CCR5 agonist described to date, and importantly, displays markedly higher HIV1 suppressive activity than all other human MIP-1alpha isoforms examined. In addition, while RANTES has been described as the most potent inhibitor of CCR5-mediated HIV entry, MIP-1alphaP was as potent as, if not more potent than, RANTES in HIV-1 suppressive assays. This property suggests that MIP-1alphaP may be of importance in controlling viral spread in HIV-infected individuals.     

Macrophage inflammatory protein-1

Macrophage inflammatory protein (MIP)-1alpha was identified 15 years ago as the first of now four members of the MIP-1 CC chemokine subfamily. These proteins termed CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CCL9/10 (MIP-1delta), and CCL15 (MIP-1gamma) according to the revised nomenclature for chemokines are produced by many cells, particularly macrophages, dendritic cells, and lymphocytes. MIP-1 proteins, which act via G-protein-coupled cell surface receptors (CCR1, 3, 5), e.g. expressed by lymphocytes and monocytes/macrophages (MPhi), are best known for their chemotactic and proinflammatory effects but can also promote homoeostasis. The encouraging results of preclinical studies in murine models of inflammation, i.e. asthma, arthritis, or multiple sclerosis, have led to the development of potent CCR3 and 5 antagonists, some of which are currently being tested in first clinical trials.