In this regard, B6 mice treated with another NK-1R antagonist, Spantide I, showed significantly improved disease outcome,
13 and the present study provides evidence that an earlier onset of apoptosis, similar to the pattern observed in naturally resistant BALB/c mice, may in part, account for the decreased pathogenesis. Consistent with earlier onset of apoptosis, mRNA expression of caspase-3 also was significantly upregulated earlier in the cornea of Spantide I, versus control treated animals. In contrast, caspase-9 levels were not different at 1 or 3 days PI, suggesting that earlier time periods would need to be tested to confirm or negate these data. The data for caspase-3, however, suggest that the protective mechanism of Spantide I treatment in B6 mice involves induction of earlier PMN apoptosis in the infected cornea, with less bystander tissue damage. Whether in B6 mice the effects of Spantide I are directly mediated via the PMNs or indirectly through Mφ regulation of PMNs was also tested. Clodronate depletion of Mφ with or without Spantide I treatment revealed that in the absence of Mφs, apoptosis was reduced or absent in the cornea. In this regard, apoptosis is an effective regulatory mechanism that can eliminate cells other than PMNs, including activated Mφs. These cells are potent immune-regulating cells that function by the release of an array of mediators, including proinflammatory cytokines such as TNF-α, IL-12, IL-1, and IL-6 and anti-inflammatory cytokines such as IL-10 and TGF-β.
14 However, unregulated and imbalanced production of Mφ-derived pro- and anti-inflammatory cytokines may exacerbate inflammation and result in enhanced disease.
30 Therefore, Mφs from both groups of mice were incubated in the presence of SP together with LPS or with LPS alone. Significantly fewer apoptotic cells were detected in cells from B6 mice in the presence of SP and LPS versus LPS alone, whereas the same combined treatment (SP and LPS) did not decrease the number of LPS-induced apoptotic Mφs in BALB/c mice. To determine the mechanism for the disparate response to SP treatment between Mφs from the two groups, we conducted comparative testing of the expression levels of the NK-1R. Although cells from both groups expressed the receptor, with a slightly weaker signal in BALB/c cells, after LPS stimulation, mRNA expression for the NK-1R was detected only in cells from B6 mice. These data suggest that the possible mechanism for the absence of the antiapoptotic effects observed in BALB/c Mφs after SP treatment may involve a low level of NK-1R expression on the cells and possible rapid depletion of the receptor on LPS stimulation. In this regard, VIPR1, the major receptor for the neuropeptide VIP, was also reported to be expressed disparately in Mφ from B6 (less) versus BALB/c (more) mice.
5 In addition, others have reported that after LPS treatment, IL-1RII, a negative regulator of the TLR/IL-1R superfamily, is downregulated in monocytes.
31 32 Our own work also showed that expression of Ig IL-1R-related molecule (SIGIRR) is significantly decreased at 12 hours PI (mRNA level) and at 1 day PI (protein level) after bacterial infection.
33 Collectively, these data, suggest that certain signaling molecules are downregulated after LPS or bacteria induced inflammation, due either to consumption of the molecule or other regulatory processes.