Anti-inflammatory effects of the nicotinergic peptides SLURP-1 and SLURP-2 on human intestinal epithelial cells and immunocytes

Abstract

A search for novel and more efficient therapeutic modalities of inflammatory bowel disease (IBD) is one of the most important tasks of contemporary medicine. The anti-inflammatory action of nicotine in IBD might be therapeutic, but its toxicity due to off-target and nonreceptor effects limited its use and prompted a search for nontoxic nicotinergic drugs. We tested the hypothesis that SLURP-1 and -2--the physiological nicotinergic substances produced by the human intestinal epithelial cells (IEC) and immunocytes--can mimic the anti-inflammatory effects of nicotine. We used human CCL-241 enterocytes, CCL-248 colonocytes, CCRF-CEM T-cells, and U937 macrophages. SLURP-1 diminished the TLR9-dependent secretion of IL-8 by CCL-241, and IFN γ-induced upregulation of ICAM-1 in both IEC types. rSLURP-2 inhibited IL-1 β-induced secretion of IL-6 and TLR4- and TLR9-dependent induction of CXCL10 and IL-8, respectively, in CCL-241. rSLURP-1 decreased production of TNFα by T-cells, downregulated IL-1 β and IL-6 secretion by macrophages, and moderately upregulated IL-10 production by both types of immunocytes. SLURP-2 downregulated TNFα and IFNγ R in T-cells and reduced IL-6 production by macrophages. Combining both SLURPs amplified their anti-inflammatory effects. Learning the pharmacology of SLURP-1 and -2 actions on enterocytes, colonocytes, T cells, and macrophages may help develop novel effective treatments of IBD.

Figure 1

Anti-inflammatory effects of rSLURP-1 and -2 on IEC. The anti-inflammatory effects of 0.01 μg/mL of rSLURP-1 (S1) and -2 (S2) on secretion of IL-6, IL-8, and CXCL10 (ELISA) and expression of ICAM-1 (QIA) by CCL-241 and CCL-248 stimulated for 16 h in a humid, 5% CO₂ incubator at a cell density of 1 × 10⁶ cells/well with 100 U/mL of IL-1β (IL-6 assay), 25 μg/mL of the TLR9 ligand E. coli DNA (IL-8), 100 ng/mL of the TLR4 ligand LPS-EK (CXCL10), or 100 U/mL of INFγ (ICAM-1) were measured as described in Materials and Methods. Some cells were exposed to S1 or S2 in the presence of 1 μg/mL of anti-SLURP-1 or -2 monoclonal antibodies (Ab). Each experiment was performed in triplicate. Asterisk = P < 0.05, compared to untreated cells. Pound sign = P < 0.05, compared to an inflammatory stimulant given alone.

Figure 2

Anti-inflammatory effects of rSLURP-1 and -2 on immunocytes. The anti-inflammatory effects of rSLURP-1 (S1) and -2 (S2), 0.01 μg/mL, on production of proinflammatory cytokines and IL-10 by the CEM stimulated with 10 μM PHA (a) and by the differentiated U937 macrophages stimulated with 200 ng/mL LPS (b) incubated for 16 h in a humid, 5% CO₂ incubator at a cell density of 1 × 10⁶ cells/well were measured by QIA, as detailed in Materials and Methods. Each experiment was performed in triplicate. Asterisk = P < 0.05, compared to intact cells. Pound sign = P < 0.05, compared to PHA or LPS given alone.