Langerhans cells (LC) play essential roles in the induction of cellular immune responses to a variety of skin-relevant antigens. Despite the fact that antigen presentation takes place primarily in draining lymph nodes, the mechanism by which LC migrate from epidermis remains uncertain. Our central hypothesis is that adhesive interaction between hyaluronan (HA) expressed abundantly on keratinocytes and CD44 expressed on activated LC mediates LC emigration. Interaction between HA (expressed on endothelial cells) and CD44 (expressed on T cells and granulocytes) is known to mediate homing of these inflammatory leukocytes. Using a phage display strategy, we have identified several 12-mer peptides that bind selectively to HA. Unlike other conventional inhibitors against CD44, our HA-binding peptides should inhibit the function of HA and, thus, provide a unique opportunity to study functional role(s) of skin-associated HA. In fact, one of the peptides, termed """"""""Peptide 1 """""""", upon local injection into mouse skin, prevented almost completely LC emigration triggered by topical application of a reactive hapten (DNFB). Injected Peptide 1 also inhibited contact sensitivity responses to DNFB at the elicitation phase. These preliminary results have led us to hypothesize further that Peptide 1 and its derivatives can be used to prevent the induction of cutaneous immune reactions in the sensitization phase and to inhibit cutaneous inflammatory reactions in the elicitation phase.
Our specific aims are: 1) To characterize the biochemical properties of HA-binding peptides. We will synthesize deletion and Ala-substituted derivatives of Peptide 1 and examine their: a) HA-binding sites, affinity, and specificity (by competitive ELISA), b) association and dissociation rates (by surface plasmon resonance analysis), and c) secondary structure (by circular dichroism analysis). 2) To determine the impact of HA-binding peptides on LC migration. Peptide 1 derivatives will be s.c. injected into BALB/c mouse skin before DNFB painting; LC emigration will then be measured by counting Ia+ epidermal cells that remain in the epidermis. 3) To study the immunological outcome of interrupted LC migration. We will determine whether injected Peptide 1 derivatives: a) inhibit hapten-induced maturation of LC, b) prevent the induction of contact hypersensitivity, and c) induce immunological tolerance. 4) To test the therapeutic efficacy of HA-binding peptides in mice. Mice that have been sensitized to DNFB (or OVA) will receive local injection of Peptide 1 derivatives immediately before challenge with DNFB (or OVA); these animals will then be examined for ear (or footpad) swelling responses to DNFB (or OVA). The proposed experiments will provide new knowledge with respect to molecular mechanisms of LC migration, functional roles of HA in skin biology, and in vivo utilities of HA inhibitors. Our studies may also lead ultimately to the development of an entirely new class of immune-suppressive and anti-inflammatory agents, which block both emigration of skin-resident leukocytes (LC) and skin-directed homing of inflammatory leukocytes.
