B6 FasL
−/− compared with WT mice also were tested to exclude the possibility that genetic discrepancies in addition to FasL were involved. For example, it was reported
25 that the number of
Qa-2 genes, a member of the nonclassical major histocompatibility complex class Ib molecules, is variable in different mouse strains, with some strains expressing all four
Qa-2 genes (B6 mice), others expressing two (BALB/cJ mice), and still others being deleted for the Qa-2 locus (C3H/HeJ and BALB/cByJ mice). Our data suggested no role for the Qa-2 locus and indicated that mutation of the
FasLgld gene itself led to worsened disease outcome. In fact, we observed a delay in the onset of apoptosis and earlier corneal perforation in B6 FasL
−/− mice. This group also had more (approximately fourfold) bacteria and PMN (MPO assay) in the cornea than did WT B6 mice, consistent with the pattern reported herein for BALB/c FasL
−/− mice. In addition, we had to test the possibility that FasL/Fas-mediated apoptosis is not essential in regulating the lifespan of PMNs, as reported by others.
26 For this we used TUNEL staining and immunostaining. The data clearly show that the cells that undergo apoptosis in the BALB/c FasL
−/− mouse cornea are PMNs, thus corroborating those findings and suggesting that in the absence of FasL, other pathways of apoptosis, though less timely, are activated and can regulate the PMN lifespan. TUNEL staining confirmed the delayed apoptotic pattern in knockout versus WT BALB/c mice, and RT-PCR combined with immunostaining correlated these data with caspase mRNA levels. The major initiator caspase in the Fas-FasL pathway, caspase 8,
6,7 which had not been tested before,
5 was downregulated in BALB/c FasL
−/−t versus WT mice after corneal infection. Knockout mice also showed delayed upregulation of both caspase 3 (BALB/c and B6 backgrounds) and caspase 9 (only BALB/c tested) compared with WT mice, and these data were similar to those shown when comparing susceptible B6 with resistant BALB/c mice.
5 Caspase 9 is mainly an initiator caspase related to the intrinsic pathway, which, on stress (e.g., infection), triggers mitochondrial release of cytochrome
c, which binds to apoptotic protease-activating factor-1, or Apaf-1. The latter recruits and activates initiator caspase 9, leading to effector caspase stimulation and cell degradation.
27–29 The data suggest that when the Fas pathway is compromised (as in the knockout mouse), other pathways, such as the intrinsic pathway, may be activated and responsible for the induction of apoptosis when the Fas pathway is not functional, as in this study.