The human cathelicidin LL-37 is expressed in neutrophils, epithelial cells, mast cells, B cells, NK cells, and γδ T cells (reviewed in 1,
28), while the detailed expression of mCRAMP is less well described. To determine whether splenic B and T cells express mCRAMP, splenocytes from C57BL/6 mice were sort-purified to obtain MZ (B220+, CD21hi, CD23low) B cells, FO (B220+, CD21int, CD23+) B cells, CD4+ and CD8+ T cells. In addition, total peritoneal lavage cells were sort-purified to obtain B1a (CD5+ Mac-1+ B220int), B1b (CD5− Mac-1+ B220int), B2 (CD5− Mac-1− B220high), and T cells (CD5+ B220−). Post-sort analysis Bioactive Compound Library manufacturer revealed greater than 95% purity for each B- and T-cell population (data not shown). Total RNA was isolated from each sort-purified cell population and RT-PCR was performed to detect Camp, CD19, CD3e, and actin mRNA. All B and T cells tested expressed Camp mRNA directly ex vivo (Fig. 1A). To determine whether B and T cells express the mCRAMP protein, total
protein was isolated from purified B and T cells and analyzed using Western blot. Figure 1B confirms the expression of the immature mCRAMP protein in the total resting B and T cells. To determine whether B and T cells regulate the expression of Camp following cell activation, total CD43− splenic B cells were sort-purified and activated with CD40L and IL-4 or IFN-γ, while selleck purified CD4+ T cells were cultured in either Th1- or Th2-inducing conditions. Real-time PCR analysis for the relative expression level of Camp, normalized to actin expression, revealed that both B and T cells increased Camp expression following activation (Fig. 1C). Interestingly, B and T cells express less Camp mRNA and mCRAMP protein Morin Hydrate relative to purified neutrophils (Fig. 1B and C). In addition, total numbers of B- and T-cell subsets as well as serum antibody levels were equivalent between
C57BL/6 and Camp−/− mice (data not shown). These data show that all B and T cells tested express Camp mRNA and mCRAMP protein, suggesting that mCRAMP has the potential to regulate B- and T-cell functions. The ability of mCRAMP to directly regulate mouse T-cell cytokine production has not been fully investigated. WT and Camp−/− naïve CD4+ T cells were sort-purified and cultured in either Th1 (anti-CD3, -CD28, and rIFN-γ) or Th2 (anti-CD3, -CD28, -IL-12, and rIL-4) inducing conditions. Under Th1-inducing conditions, WT and Camp−/− T cells expressed equivalent amounts of IFN-γ mRNA (Fig. 2A), equivalent numbers of IFN-γ+ cells (Fig. 2B), and equivalent IFN-γ mean fluorescent intensity (MFI) (Fig. 2C). In contrast, Camp−/− T cells cultured under Th2-inducing conditions expressed more IL-4 mRNA (Fig. 2D), more IL-4+ cells (Fig. 2E), and equivalent IL-4 MFI (Fig. 2F).