Patricia Menten

Macrophage inflammatory protein-1

Macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta are highly related members of the CC chemokine subfamily. Despite their structural similarities, MIP-1alpha and MIP-1beta show diverging signaling capacities. Depending on the MIP-1 subtype and its NH(2)-terminal processing, one or more of the CC chemokine receptors CCR1, CCR2, CCR3 and CCR5 are recognized. Since both human MIP-1alpha subtypes (LD78alpha and LD78beta) and MIP-1beta signal through CCR5, the major co-receptor for M-tropic HIV-1 strains, these chemokines are capable of inhibiting HIV-1 infection in susceptible cells. In this review, different aspects of human and mouse MIP-1alpha and MIP-1beta are discussed, including their protein and gene structures, their regulated production, their receptor usage and biological activities and their role in several pathologies including HIV-1 infection.

Natural truncation of RANTES abolishes signaling through the CC chemokine receptors CCR1 and CCR3, impairs its chemotactic potency and generates a CC chemokine inhibitor

Selective leukocyte trafficking towards sites of inflammation is mediated by chemokines. RANTES is a CC chemokine that attracts lymphocytes, monocytes, dendritic cells, eosinophils, basophils and NK cells. A natural form of human RANTES lacking two N‐terminal residues was isolated from stimulated sarcoma cells, fibroblasts, and leukocytes. RANTES(3 – 68) showed a more than tenfold reduction in chemotactic potency for monocytes and eosinophils. To elucidate the mechanism involved, receptor recognition studies were performed. In cells transfected with the CC chemokine receptor (CCR) 5, the major co‐receptor for macrophage‐tropic HIV‐1 strains, RANTES(3 – 68) mobilized calcium and desensitized RANTES(1 – 68)‐induced calcium fluxes equally well as RANTES(1 – 68). However, RANTES(3 – 68) was ineffective on CCR1 and CCR3 transfectants. The reduced potency of natural RANTES(3 – 68) by selective loss of receptor‐activating characteristics was confirmed with recombinant RANTES(3 – 68). In chemotaxis assays using monocytic cells, RANTES(3 – 68) inhibited RANTES(1 – 68), macrophage inflammatory protein‐1α (MIP‐1α), MIP‐1β or monocyte chemotactic protein‐3 (MCP‐3), but not MCP‐1‐ or MCP‐2‐induced chemotaxis. Thus, a minor post‐translational modification has a remarkable impact on the biological activities of RANTES and a pathophysiologically induced change in the relative amounts of intact and truncated RANTES might affect the outcome of inflammation or HIV infection.

The LD78beta isoform of MIP-1alpha is the most potent CCR5 agonist and HIV-1-inhibiting chemokine.

LD78alpha and LD78beta are 2 highly related nonallelic genes that code for different isoforms of the human CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). Two molecular forms of natural LD78beta (7.778 and 7.793 kDa) were identified from conditioned media of stimulated peripheral blood mononuclear cells. Although LD78alpha and LD78beta only differ in 3 amino acids, both LD78beta variants were 100-fold more potent chemoattractants for mouse lymphocytes than was LD78alpha. On the contrary, LD78beta was only 2-fold more efficient than LD78alpha in chemoattracting human lymphocytes and monocytes. Using CC chemokine receptor-transfected cells, both molecular forms of LD78beta proved to be much more potent than LD78alpha in inducing an intracellular calcium rise through CCR5. Compared with LD78alpha and RANTES, this preferential binding of LD78beta to CCR5 resulted in a 10- to 50-fold higher potency in inhibiting infection of peripheral blood mononuclear cells by CCR5-using (R5) HIV-1 strains. To date, LD78beta is the most potent chemokine for inhibiting HIV-1 infection, and can be considered as a potentially important drug candidate for the treatment of infection with R5 HIV-1 strains.

Diverging binding capacities of natural LD78beta isoforms of macrophage inflammatory protein-1alpha to the CC chemokine receptors 1,3 and 5 affect their anti-HIV-1 activity and chemotactic potencies for neutrophils and eosinophils

Recently, the LD78β isoform of the CC chemokine macrophage inflammatory protein (MIP)‐1α was shown to efficiently chemoattract lymphocytes and monocytes and to inhibit infection of mononuclear cells by R5 HIV‐1 strains. We have now demonstrated that after cleavage of the NH2‐terminal Ala‐Pro dipeptide by CD26, LD78β(3 – 70) became the most potent chemokine blocking HIV‐1. LD78β(3 – 70) competed tenfold more efficiently than LD78β(1 – 70) with [125I] RANTES for binding to the CC chemokine receptors CCR5 and CCR1. Contrary to LD78α, LD78β(1 – 70) at 30 ng / ml efficiently competed with [125I] RANTES for binding to CCR3 and mobilized calcium in CCR3 transfectants, whereas LD78β(3 – 70) showed a 30‐fold decrease in CCR3 affinity compared to LD78β(1 – 70). This demonstrates the importance of the penultimate proline in LD78β(1 – 70) for CCR3 recognition. Both LD78β isoforms efficiently chemoattracted eosinophils from responsive donors. In contrast, only the CCR3 agonist LD78β(1 – 70) and not LD78β(3 – 70), induced calcium increases in eosinophils with low levels of CCR1. In responder neutrophils, LD78β(3 – 70) elicited calcium fluxes at a 30‐fold lower dose (10 ng / ml) compared to intact LD78β and LD78α, whereas the three MIP‐1α isoforms were equipotent neutrophil chemoattractants. Taken together, both LD78β isoforms are potent HIV‐1 inhibitors (CCR5) and activators for neutrophils (CCR1) and eosinophils (CCR1, CCR3), affecting infection and inflammation.

The CXC chemokine GCP-2/CXCL6 is predominantly induced in mesenchymal cells by interleukin-1beta and is down-regulated by interferon-gamma: comparison with interleukin-8/CXCL8.

Human granulocyte chemotactic protein-2 (GCP-2)/CXCL6 is a CXC chemokine that functionally uses both of the IL-8/CXCL8 receptors to chemoattract neutrophils but that is structurally most related to epithelial cell–derived neutrophil attractant-78 (ENA-78)/CXCL5. This study provides the first evidence that GCP-2 protein is, compared with IL-8, weakly produced by some sarcoma, but less by carcinoma cells, and is tightly regulated in normal mesenchymal cells. IL-1β was the predominant GCP-2 inducer in fibroblasts, chondrocytes, and endothelial cells, whereas IL-8 was equally well up-regulated in these cells by TNF-α, measles virus, or double-stranded RNA (dsRNA). In contrast, lipopolysaccharide (LPS) was a relatively better stimulus for GCP-2 versus IL-8 in fibroblasts. IFN-γ down-regulated the GCP-2 production in fibroblasts induced by IL-1β, TNF-α, LPS, or dsRNA. The kinetics of GCP-2 induction by IL-1β, LPS, or dsRNA in fibroblasts differed from those of IL-8. Freshly isolated peripheral blood mononuclear leukocytes, which are a good source of IL-8 and ENA-78, failed to produce GCP-2. However, lung macrophages and blood monocyte–derived macrophages produced GCP-2 in response to LPS. Quantitatively, secretion of GCP-2 always remained inferior to that of IL-8, despite the fact that the ELISA recognized all posttranslationally modified GCP-2 isoforms. The expression of GCP-2 was confirmed in vivo by immunohistochemistry. The patterns of producer cell types, inducers and kinetics and the quantities of GCP-2 produced, suggest a unique role for GCP-2 in physiologic and pathologic processes.

Regulated production and molecular diversity of human liver and activation-regulated chemokine/macrophage inflammatory protein-3 alpha from normal and transformed cells.

Liver and activation-regulated chemokine (LARC), also designated macrophage inflammatory protein-3α (MIP-3α), Exodus, or CCL20, is a C-C chemokine that attracts immature dendritic cells and memory T lymphocytes, both expressing CCR6. Depending on the cell type, this chemokine was found to be inducible by cytokines (IL-1β) and by bacterial, viral, or plant products (including LPS, dsRNA, and PMA) as measured by a specific ELISA. Although coinduced with monocyte chemotactic protein-1 (MCP-1) and IL-8 by dsRNA, measles virus, and IL-1β in diploid fibroblasts, leukocytes produced LARC/MIP-3α only in response to LPS. However, in myelomonocytic THP-1 cells LARC/MIP-3α was better induced by phorbol ester, whereas in HEp-2 epidermal carcinoma cells IL-1β was the superior inducer. The production levels of LARC/MIP-3α (1–10 ng/ml) were, on the average, 10- to 100-fold lower than those of IL-8 and MCP-1, but were comparable to those of other less abundantly secreted chemokines. Natural LARC/MIP-3α protein isolated from stimulated leukocytes or tumor cell lines showed molecular diversity, in that NH2- and COOH-terminally truncated forms were purified and identified by amino acid sequence analysis and mass spectrometry. In contrast to other chemokines, including MCP-1 and IL-8, the natural processing did not affect the calcium-mobilizing capacity of LARC/MIP-3α through its receptor CCR6. Furthermore, truncated natural LARC/MIP-3α isoforms were equally chemotactic for lymphocytes as intact rLARC/MIP-3α. It is concluded that in addition to its role in homeostatic trafficking of leukocytes, LARC/MIP-3α can function as an inflammatory chemokine during host defense.

The LD78β Isoform of MIP-1α Is the Most Potent CC-Chemokine in Inhibiting CCR5-Dependent Human Immunodeficiency Virus Type 1 Replication in Human Macrophages

ABSTRACT The CC-chemokines RANTES, macrophage inflammatory protein 1α (MIP-1α), and MIP-1β are natural ligands for the CC-chemokine receptor CCR5. MIP-1α, also known as LD78α, has an isoform, LD78β, which was identified as the product of a nonallelic gene. The two isoforms differ in only 3 amino acids. LD78β was recently reported to be a much more potent CCR5 agonist than LD78α and RANTES in inducing intracellular Ca2+ signaling and chemotaxis. CCR5 is expressed by human monocytes/macrophages (M/M) and represents an important coreceptor for macrophage-tropic, CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains to infect the cells. We compared the antiviral activities of LD78β and the other CC-chemokines in M/M. LD78β at 100 ng/ml almost completely blocked HIV-1 replication, while at the same concentration LD78α had only weak antiviral activity. Moreover, when HIV-1 infection in M/M was monitored by a flow cytometric analysis using p24 antigen intracellular staining, LD78β proved to be the most antivirally active of the chemokines. RANTES, once described as the most potent chemokine in inhibiting R5 HIV-1 infection, was found to be considerably less active than LD78β. LD78β strongly downregulated CCR5 expression in M/M, thereby explaining its potent antiviral activity.

Role of the autocrine chemokines MIP-1α and MIP-1β in the metastatic behavior of murine T cell lymphoma

The ESb‐MP T‐cell line is a highly malignant murine lymphoma, which preferentially metastasizes toward the kidney. This could be a result of the local production of monocyte chemoattractant protein‐1 (MCP‐1) and regulated on activation, normal T expressed and secreted (RANTES), which are chemotactic for ESb‐MP cells. Here, we demonstrate that ESb‐MP cells are already responsive to the chemotactic activity of macrophage inflammatory protein‐1α (MIP‐1α) and MIP‐1β from 1 ng/ml onward. Moreover, upon stimulation with lipopolysaccharide (LPS) or virus, ESb‐MP cells themselves produce significant amounts of MIP‐1 (∼200 ng/ml). Indeed, the major autocrine chemoattractants, isolated from ESb‐MP cells, were intact MIP‐1α and MIP‐1β. Pretreatment with LPS or addition of MIP‐1 inhibited the in vitro migration of ESb‐MP cells toward various chemokines. Moreover, compared with untreated lymphoma cells, LPS‐treated cells produced significantly less metastasis in mice. The results represented here suggest that the role of chemokines in attracting tumor cells at secondary sites depends on a balance between autocrine‐produced and tissue‐derived chemokines. This delicate balance should be considered in the design of antichemokine strategies in different tumor types.