Background-Pollens are important triggers for asthma but the mechanism of sensitisation to their proteins remains poorly understood. The intrinsic protease activity of some allergens may contribute to sensitisation by disrupting the integrity of the airway epithelial barrier. Pollens release a variety of enzymes, including proteases, upon hydration. The hypothesis that such enzymes might be able to damage airway epithelial cells was therefore tested. Methods-DiVusates from pollens of Lolium perenne (ryegrass), Poa pratensis (Kentucky bluegrass), Acacia longifolia (Sydney golden wattle), or Casuarina distyla (she-oak) were incubated with mouse tracheal epithelial cells in culture and cellular detachment was quantified using a methylene blue dye binding assay. Results-DiVusates prepared using 100 mg/ml of pollen caused detachment of 30-90% of airway epithelial cells in separate experiments. Within each experiment comparable detachment was observed with all diVusates tested, although total protein in the diVusates varied markedly between species. Viability of the cells recovered after exposure to Acacia diVusate was higher than after detachment by exposure to Lolium diVusate. Cellular detachment by all of the diVusates could be almost completely inhibited by addition of 10% serum. Aprotinin, an inhibitor of serine proteases, partially blocked activity in diVusates of Lolium pollen but not of Acacia pollen. In contrast, 1 -protease inhibitor and secretory leucocyte protease inhibitor (SLPI) were not able to block the activity of either diVusate at concentrations which inhibited cellular detachment by trypsin. Conclusions-Proteases released by pollens are able to cause detachment of airway epithelial cells from their substratum in vitro and may not be eVectively inhibited by endogenous antiproteases.

Chemokines mediate trafficking of leukocytes to sites of inflammation and immune responses through activation of G protein-coupled receptors, which thereby provide appealing targets for novel anti-inflammatory agents. Vasoactive intestinal peptide (VIP) is an immunosuppressive neurotransmitter. We show that VIP inhibited the function of chemokine receptors on monocytes and CD4+ T lymphocytes, with impaired chemotaxis and calcium flux in response to the cognate chemokine ligands CXCL12, CCL3, CCL4, and CCL5. This was mediated by VIP receptor type 1 and was not caused by chemokine receptor internalization. However, VIP caused dose-dependent phosphorylation of the chemokine receptor CCR5. This trans-deactivation process was studied in a murine model of delayed-type hypersensitivity: continuous infusion of VIP resulted in significant abrogation of monocyte and lymphocyte infiltration. Circulating mononuclear cells from VIP-infused mice were unable to respond to chemokines. VIP may provide a novel approach to treatment of inflammatory diseases through inhibition of chemokine-dependent leukocyte recruitment.

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Mitogens of the EGF family may play an important role in regulating the proliferation of airway epithelial cells (AEC). We examined the production of autocrine mitogenic activity by mouse AEC cultured from explants of tracheal tissue. DNA synthesis by growth-arrested AEC was stimulated by conditioned media from cells maintained in serum-free culture without exogenous growth factors. The mitogenic activity was blocked by a specific inhibitor of the EGF receptor tyrosine kinase. Furthermore, conditioned media from AEC contained molecular species that could compete with radiolabeled EGF in a receptor binding assay. However, mitogenic activity was not blocked by neutralizing antibodies to EGF or to transforming growth factor-alpha, but was partly inhibited by co-incubation with heparin, suggesting that it might be due to a heparin-binding member of the EGF family. The activity was potentiated by co-incubation with IGF-1, analogous to the potentiation by IGF-1 of the mitogenic activity of EGF for AEC. Moreover, the autocrine mitogen produced by AEC exhibited cooperative interaction with the mitogenic activity in conditioned media from growth factor-deprived mouse lung fibroblasts, consistent with the hypothesis that interactions with mesenchymal cells could influence the proliferation of AEC in vivo.