This study demonstrates the important role of PLA2 in killing S. aureus in the rabbit tear film and shows that this activity significantly declines with age, increases during sleep, and subsequently declines after periods of sleep. Furthermore, the results show that the PLA2 activity in the tears and aqueous humor increases in parallel with PMN concentrations (MPO activity) and protein increases in the tissue during S. aureus keratitis.
Previous findings by Moreau et al.
14 demonstrated the ability of PLA
2 in rabbit tears to kill
S. aureus. The data presented herein show the bactericidal role of this enzyme in the tear film and also indicate that the bactericidal activity of this enzyme decreases with the age of the rabbit. The highest bactericidal activity was clearly observed in those tears collected from young rabbits, whereas the bactericidal activity declined significantly in those tears collected from aged rabbits. The results of the bactericidal assay correlated with those results obtained from the radioactive assay specific for PLA
2, so that enzyme activity declined significantly with age. Saari et al.
39 demonstrated similar findings with tears collected from human subjects, demonstrating a decrease in PLA
2 concentration that occurred with an increase in age. These findings suggest that loss of this important ocular host defense could render the corneas of aged individuals more susceptible to
S. aureus infection than the corneas of younger individuals.
The effects of the sleep cycle on the activity of PLA
2 in rabbit tears were also determined. Peaks in PLA
2 activity were noted in tears collected from rabbits in deep sleep, with a steady decline in activity noted as the day progressed, when rabbits were awake and active. These findings correlate with studies that suggest that the composition of the tear film varies between open-eye tears and closed-eye tears.
40 The normal composition of tears that are collected from open eyes is primarily lysozyme, lactoferrin, lipocalin, and secretory IgA (sIgA).
40 However, during sleep, there is a decrease in tear flow, and the concentration of total tear protein increases.
41 The composition of the tear film is altered, so that sIgA concentrations increase, serum proteins (tear albumin, fibronectin, and complement) increase, and there is a large recruitment of PMNs to the tear film.
40 41 42 43 Because neutrophils are an established source of PLA
2,
44 45 their influx into the tear film could serve as an important source of PLA
2 during sleep.
The role of PLA
2 was also analyzed during the progression of
S. aureus keratitis. Activity of PLA
2 increased substantially, as measured in the aqueous humor and tear film collected from rabbits as the infection progressed. Increased PLA
2 activity also correlated with an increase in total protein concentration in these two fluids, although some of the increases in total protein could be due to increased colony-forming units at the later time points. Inflammation that is characteristic of keratitis in the rabbit model includes chemosis, injection, and accumulation of pus on the corneal surface.
34 46 Soluble bacterial products, such as toxins,
47 mediate tissue damage and induce host chemotactic agents, such as complement and cytokines, that are known mediators of ocular inflammation.
47 48 In addition, inflammation may be due in part to the release of arachidonic acid after digestion of the bacterial inoculum by PLA
2.
The increase in PLA
2 activity in infected rabbit eyes probably reflects a common host response to changes in the ocular environment. Song et al.
49 have demonstrated increased PLA
2 activity in the eyes of patients with chronic blepharitis. Tears collected from patients with blepharitis contained twice the PLA
2 activity of those of normal individuals.
49 In addition, Aho et al.
50 found a similar two-fold increase in PLA
2 activity in the eyes of patients with keratoconjunctivitis sicca.
The parallel increase in MPO and PLA
2 activity during infection suggests that PMNs are an important source of PLA
2 in the tear film, particularly in response to
S. aureus keratitis. Sloop et al.
36 have demonstrated that neutrophils in the tear film are derived in part from the eyelid, and that the eyelid-derived PMNs far exceed those that migrate into the cornea from the limbus during
Staphylococcus keratitis. The present study confirms this point, in that the MPO activity of the tear film was several times higher than that of the corresponding corneal homogenate. These results are also supported by other studies that have demonstrated that neutrophils from the tear film are involved in inflammation of the cornea.
36 51 52 In addition, one mechanism proposed by Song et al.
49 for the increased PLA
2 activity observed in patients with chronic blepharitis suggests that PMNs responding to bacterial inflammatory factors migrate into the tear film and then release quantities of PLA
2.
In summary, this study has demonstrated factors that affect the host defense of the ocular tear film by altering the activity of phospholipase A2. Factors that affect host defense include age and the sleep cycle, as well as the progression of keratitis mediated by S. aureus.
The authors thank Julian Reed and Quentin Booker for technical assistance with the research.