Induction of anti-chlamydial mucosal immunity by transcutaneous immunization is enhanced by topical application of GM-CSF

Transcutaneous immunization (TCI) involves the direct application of antigen plus adjuvant to skin, taking advantage of the large numbers of Langerhans cells and other resident skin dendritic cells, that process antigen then migrate to draining lymph nodes where immune responses are initiated. We have used this form of immunization to protect mice against genital tract and respiratory tract chlamydial infection. Protection was associated with local antibody responses in the vagina, uterus and lung as well as strong Th1 responses in the lymph nodes draining the reproductive tract and lungs respectively. In this study we show that topical application of GM-CSF to skin enhances the numbers and activation status of epidermal dendritic cells. Topical application of GM-CSF also increased the immune responses elicited by TCI. GM-CSF supplementation greatly increased cytokine (IFNgamma and IL-4) gene expression in lymph node and splenic cells compared to cells from animals immunized without GM-CSF. IgG responses in serum, uterine lavage and bronchoalveolar lavage and IgA responses in vaginal lavage were also increased by topical application of GM-CSF. The studies show that TCI induces protection against genital and respiratory tract chlamydial infections and that topical application of cytokines such as GM-CSF can enhance TCI-induced antibody and cell-mediated immunity.

Gonadotropin-induced ovarian cancer cell migration and proliferation require extracellular signal-regulated kinase 1/2 activation regulated by calcium and protein kinase Cδ

The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKCδ inhibitor rottlerin, and downregulation of PKCδ was inhibited by small interfering RNA (siRNA), highlighting the importance of PKCδ in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKCδ siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKCδ-dependent manner.