Substance P delays apoptosis, enhancing keratitis after Pseudomonas aeruginosa infection.

Purpose: Apoptosis was examined after Pseudomonas aeruginosa corneal infection in C57BL/6 (B6, susceptible) and BALB/c (resistant) mice.

Methods: TUNEL staining, real-time RT-PCR, polymorphonuclear neutrophils (PMNs) and macrophage (Mphi) depletion, and immunostaining were used.

Results: Intense TUNEL staining was seen in BALB/c versus B6 cornea at 1 versus 3 days after infection (PI) and correlated with mRNA levels for caspase-3. TUNEL staining (with or without PMN depletion) and PMN immunostaining revealed the PMN as the major apoptotic cell for both groups. Next, B6 mice with high corneal levels of the antiapoptosis neuropeptide, substance P (SP), were treated with the SP antagonist, Spantide I (with/without Mphi depletion), resulting in earlier apoptosis and diminished disease only when M(phi)s were present. SP interactions with M(phi)s were explored further by eliciting cells from both groups and stimulating them with lipopolysaccharide (LPS), with or without SP. LPS with SP treatment decreased the number of apoptotic M(phi)s in B6 but not BALB/c mice and correlated with reduced mRNA expression of NK-1R (major SP receptor) on BALB/c cells. In addition, mRNA expression for IL-12 was upregulated in LPS-stimulated B6 M(phi)s, although cells from BALB/c mice expressed more IL-10.

Conclusions: These studies provide evidence that PMN apoptosis is delayed in the cornea of B6 versus BALB/c mice after bacterial infection; that in B6 mice, blocking SP interaction with the NK-1R promotes earlier apoptosis and improves disease outcome; that M(phi)s regulate PMN apoptosis; and that M(phi)s from B6 versus BALB/c mice differ in expression of the NK-1R and cytokines produced after LPS challenge.