In the present study, topical Tβ
4 treatment promoted corneal clarity in a BALB/c model of alkali injury. These novel findings extend those of our previous studies of topical Tβ
4 treatment, to demonstrate quantitatively that the healing effect of Tβ
4 on the cornea is associated with a decrease in PMN infiltration, downregulation of the chemokines MIP-2 and KC, and a decrease in the expression of three major MMPs: the gelatinases (MMP-2 and -9) and MT6-MMP. The relationship of corneal inflammation (chemokine expression), MMP expression, and wound healing with Tβ
4 has not been studied previously. Because the mechanisms of action of Tβ
4 in wound healing are not well defined, our data shed new light on possible pathways that may be modulated during repair. A major component that influences visual outcome and ocular morbidity after chemical burn is the severity of the host inflammatory response. PMN infiltration during corneal ulceration and into injured corneal tissue during wound repair is a well-recognized phenomenon.
30 31 Because most of the ensuing ocular complications stem from the massive PMN infiltration of the stroma,
2 therapeutic strategies that limit PMN infiltration of the corneal stroma may prevent and ameliorate the ocular morbidity that follows inflammation-mediated damage. In this study, Tβ
4 treatment significantly inhibited the expression of two murine chemokines, KC and MIP-2, suggesting that Tβ
4 inhibits PMN infiltration by downregulating these proinflammatory mediators. Regarding the KC RT-PCR and ELISA data, whereas similar expression trends were noted at days 3 and 7 PI, there appeared to be a slight increase in the KC protein levels on day 7 in the Tβ
4-treated corneas. Although the graphic representation of the mRNA
(Fig. 5A)shows a slight decrease between days 3 and 7 PI, the difference is not statistically significant. It is possible that in the individual corneas assayed, posttranscriptional and posttranslational modifications of proteins may occur, and that may explain the discrepancy in the protein levels for these days
(Fig. 5B) . After corneal alkali burn, our results show that the expression of KC and MIP-2 may be responsible for triggering PMN influx into the cornea to initiate an intense inflammatory cascade. These molecules are functionally homologous to human IL-8 and exhibit potent PMN chemotactic activity when mediating neutrophil recruitment in response to tissue injury and infection.
32 33 34 35 We monitored the kinetics of PMN recruitment to the cornea after alkali injury by measuring MPO activity levels, with and without Tβ
4 treatment. Our data show that Tβ
4 treatment reduced corneal MPO levels, indicating that Tβ
4 is a potent inhibitor of PMN infiltration after corneal injury, consistent with reports of Tβ
4 as an anti-inflammatory agent and previous studies showing that Tβ
4 inhibits PMN migration in vivo.
23 36 However, in vitro studies showed that Tβ
4 had no effect on PMN migration
37 suggesting that the anti-PMN activity of Tβ
4 in vivo may involve indirect effects on PMNs, possibly by downregulation of chemokine production as shown here. Whether Tβ
4 exerts its effects on PMNs directly or indirectly is an intriguing question. As the anti-inflammatory properties of Tβ
4 are beginning to be elucidated, other recent studies have shown the activation-responsive expression of the lymph-specific form of Tβ
4 may be one mechanism by which dendritic epidermal T cells and possibly other intraepithelial lymphocytes downregulate local inflammation.
38 In a separate study, Tβ
4 lowered circulating levels of inflammatory cytokines and intermediates after lipopolysaccharide (LPS) administration in vivo. In addition, Tβ
4 levels rapidly disappeared in the blood after LPS administration or during septic shock, suggesting that Tβ
4 may be involved in early events leading to activation of the inflammatory cascade and ultimately the clinical sequelae of sepsis.
39 Thus, the anti-inflammatory effects found with topical Tβ
4 treatment in the current study may have clinical relevance as a novel therapeutic agent for treating corneal inflammation.