Atherosclerosis is characterized by chronic inflammation of the arterial wall due to chemokine-driven mononuclear cell recruitment. Activated platelets can synergize with chemokines to exacerbate atherogenesis; for example, by deposition of the chemokines platelet factor-4 (PF4, also known as CXCL4) and RANTES (CCL5), triggering monocyte arrest on inflamed endothelium. Homo-oligomerization is required for the recruitment functions of CCL5, and chemokine heteromerization has more recently emerged as an additional regulatory mechanism, as evidenced by a mutual modulation of CXCL8 and CXCL4 activities and by enhanced monocyte arrest resulting from CCL5-CXCL4 interactions. The CCL5 antagonist Met-RANTES reduces diet-induced atherosclerosis; however, CCL5 antagonism may not be therapeutically feasible, as suggested by studies using Ccl5-deficient mice which imply that direct CCL5 blockade would severely compromise systemic immune responses, delay macrophage-mediated viral clearance and impair normal T cell functions. Here we determined structural features of CCL5-CXCL4 heteromers and designed stable peptide inhibitors that specifically disrupt proinflammatory CCL5-CXCL4 interactions, thereby attenuating monocyte recruitment and reducing atherosclerosis without the aforementioned side effects. These results establish the in vivo relevance of chemokine heteromers and show the potential of targeting heteromer formation to achieve therapeutic effects.

In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/ CCL2, RANTES/CCL5, and MIP-1␤/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAGinduced oligomerization beyond retention of chemokines at the site of production.

Specific immunotherapy is an efficient treatment for patients suffering from type I allergy. The mechanisms underlying successful immunotherapy are assumed to operate at the level of T helper cells, leading to a modulation of the immune response to allergens. During immunotherapy, increasing doses of allergens are given on a regular basis, and the beneficial effects for the patient depend on the concentration of allergen used. On the other hand, the risk of IgE-mediated anaphylactic side effects also increase with the amount of allergen applied per injection. Therefore, we have proposed the use of hypoallergenic (low IgE binding activity) forms of allergens for immunotherapy. We evaluated by site-directed mutagenesis the contributions of individual amino acid residues/positions for IgE binding to Bet v 1, the major allergen of birch pollen. We found that IgE binding to Bet v 1 depended on at least six amino acid residues/positions. Immunoblot analyses and inhibition experiments showed that the multiple-point Bet v 1 mutant exhibited extremely low reactivity with serum IgE from birch pollen-allergic patients. In vivo (skin prick) tests showed that the potency of the multiple-point mutant to induce typical urticarial type I reactions in pollen-allergic patients was significantly lower than for wild-type Bet v 1. Proliferation assays of allergen-specific T cell clones demonstrated that these six amino acid exchanges in the Bet v 1 sequence did not influence T cell recognition. Thus, the Bet v 1 six-point mutant displayed significantly reduced IgE binding activity, but conserved T cell activating capacity, which is necessary for immunomodulation. The approach described here may be generally applied to produce allergen variants to be used in a safe therapy form of immediate-type allergies.

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Background: Chemokines, such as CXCL8 and CXCL9, drive leukocyte migration to an inflammation site. Results: CXCL9(74 -103), derived from CXCL9, lacks leukocyte-attracting activity but competes with CXCL8 for GAG binding and inhibits neutrophil migration in two murine acute inflammation models. Conclusion: Through inhibition of chemokine-GAG interaction, CXCL9(74 -103) blocks neutrophil migration. Significance: CXCL9(74 -103) may be a lead molecule for development of anti-inflammatory agents.