INTRODUCTION

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Because the normal cornea lacks blood vessels, many of its host defense needs are supplied by the tear film. Alexander Fleming reported the presence of lysozyme in human tears over 75 years ago and described its ability to lyse and kill Micrococcus leisodeikticus (7, 8). Later investigators reported that human tears contained a nonlysozyme antistaphylococcal factor that also killed M. leisodeikticus (10, 43) Ford, et al. have advanced (9) and others have challenged (17, 38) the possibility that the additional bactericidal factor is "beta-lysin"an ill-defined, heat-stable antimicrobial molecule reportedly present in platelets and plasma (4). In the absence of more precise knowledge, contemporary ophthalmology texts (23) attribute the antimicrobial properties of tears to their high concentrations of lysozyme, lactoferrin, and immunoglobulin A (IgA) (20). The experiments described below demonstrate that this conventional belief requires modification and that secretory phospholipase A₂ (sPLA₂) is an important host defense molecule in the external eye.

	MATERIALS AND METHODS

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Tear collection. Tears were collected from six healthy donors, under a protocol approved by the UCLA Institutional Review Board. After approximately 5 µl of basal, unstimulated tears had been collected over 2 min into a calibrated 5-µl pipette (Accupette; Baxter, McGaw Park, Ill.), the donors were briefly exposed to the vapors of freshly minced onions, and between 100 to 250 µl of stimulated tears was collected over the next 5 to 10 min. These tear specimens were stored at 20°C until tested.

Bacteria. Six strains of Staphylococcus aureus were used. S. aureus GM-1 and 67395 were ocular isolates obtained from the UCLA Clinical Laboratory. The GM-1 strain was gentamicin resistant. Micrococcus luteus ATCC 4698 (previously called Micrococcus leisodeikticus) and a methicillin-resistant S. aureus (MRSA) strain, ATCC 33591, were purchased from the American Type Culture Collection (Rockville, Md.). Six strains (three MRSA, one S. epidermidis, one group B streptococcus, and one vancomycin-resistant Enterococcus faecium strain [VREF 94.132]) were clinical isolates obtained from the UCLA Clinical Microbiology Laboratory. Bacillus subtilis was a laboratory reference strain, and Listeria monocytogenes EGD was a gift from Pieter Hiemstra.

Artificial tear solution. Many of our studies were performed with an artificial tear solution (ATS), a balanced salt solution whose composition simulated that of normal human tears (1). The composition of ATS was 124 mM Na⁺, 133 mM Cl, 24 mM HCO₃, 30 mM K⁺, 0.7 mM Mg²⁺, 0.7 mM Ca²⁺, 0.35 mM glucose, 4.5 mM urea, 3.5 mM lactate, and 0.2 mM pyruvate.

Colony counting assays. Mid-logarithmic-phase bacteria were washed with ATS containing 3 mg of Trypticase soy broth powder ml¹ and adjusted to contain approximately 10⁷ CFU ml¹. Samples of pooled tears, purified tear sPLA₂, lysozyme, or lactoferrin were serially diluted in ATS that had been supplemented with 0.1% bovine serum albumin (ATS-BSA). Bacteria (10 µl) were mixed with 90 µl of the pooled tear or purified protein samples, so that the final volume (100 µl) of ATS contained 0.3 mg of Trypticase soy broth powder ml¹. After 15 min, 1 h, or 3 h, aliquots were transferred to Trypticase soy agar plates with a Spiral Plater (SpiralTech, Rockville, Md.), and surviving colonies were counted after overnight incubation at 37°C.

Radial diffusion assays. An extensive description of the assay recently appeared elsewhere (40). The principal modification introduced for this study involved incorporating ATS and 0.1 mg of albumin ml¹ (Sigma A-7030) into the underlay gels. The ATS simulated a lachrymal environment, whereas the albumin minimized nonspecific adsorption of sPLA₂ to agarose, especially when ultralow (nanogram) quantities were tested. The underlay contained 4 × 10⁶ bacterial CFU dispersed in 10 ml of a gel that contained full-strength ATS, 1% agarose, 0.3 mg of Trypticase soy broth powder ml¹, and 0.01% BSA. To prepare bacteria in the logarithmic growth phase, overnight cultures in Trypticase soy broth were subcultured in fresh broth for 2.5 h. These organisms were washed twice with ATS and adjusted to the desired concentrations, based on optical density at 620 nm measurements. Serial "half-log" (i.e., 3.16-fold) dilutions of purified PLA₂ were prepared in ATS-BSA.

PLA₂ and lysozyme purification. Tears collected from different donors were pooled and subjected to reverse-phase high-performance liquid chromatography on a Vydac C₁₈ column (10 by 250 mm) (The Separations Group, Hesperia, Calif.) with a linear gradient of 0 to 60% acetonitrile that contained 0.1% trifluoroacetic acid. Each fraction was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by the sPLA₂ enzyme assay described below. Fractions with phospholipase activity were combined and rechromatographed on a Vydac C₁₈ column (4.6 by 250 mm) with a linear gradient of acetonitrile that contained 0.13% heptafluorobutyric acid (HFBA) as the ion-pairing agent. To prepare highly purified human tear lysozyme, we subjected pooled tears to preparative acid urea-PAGE (13) and performed the two-stage RP-HPLC procedure described above for PLA₂.

Enzymatic assay of PLA₂. PLA₂ activity was measured essentially as described by Elsbach and Weiss (5). Briefly, E. coli ML-35 was labeled with [¹⁴C]oleic acid (50 mCi mmol¹ [NEN/Dupont]), autoclaved, and used as the substrate. Each sample was mixed with 50,000 cpm of ¹⁴C-labeled E. coli, equivalent to 2.5 × 10⁸ bacteria (adjusted by addition of nonradioactive autoclaved E. coli) in a 250-µl volume that contained 250 mM Tris, 10 mM CaCl₂, and 1 mg of BSA per ml (pH 9.5). After a 1-h incubation in a 37°C shaking water bath, 100 µl of 2 N HCl was added to stop the reaction, and the product (free fatty acids and lysophospholipids) was trapped by adding 100 µl of 20 mg of fatty acid-free BSA ml¹. The mixture was kept at 4°C for 30 min and centrifuged at 10,000 × g for 5 min. The pellet was washed twice with 0.1% acetic acid. The supernatant and washings were combined, and their PLA₂ activity was measured by liquid scintillation counting and converted to arbitrary units (AU [1 AU = 1% release of ¹⁴C]).

Immunological assay of PLA₂. Serially diluted tears and purified tear PLA₂ standards were prepared in 5 µl of ATS containing 0.1% BSA. The standards contained 15, 12.5, 10, 7.5, and 5 ng of sPLA₂ per 5 µl. One hundred fifty microliters of Tris-buffered saline (20 mM Tris [pH 7.5], 500 mM NaCl) was added to each well of a Bio-Dot SF microfiltration apparatus (Bio-Rad, Hercules, Calif.). Five microliters of the purified tear PLA₂ standards or of a dilution series of tears was added, followed by another 100 µl of Tris-buffered saline. The samples were blotted onto a Hybond-ECL (enhanced chemiluminescence) nitrocellulose membrane (Amersham, Arlington Heights, Ill.) at unit gravity. ECL assay reagents were purchased from Amersham, and ECL Western blotting was performed by strictly following the manufacturer's protocols. A murine monoclonal IgG1 antibody to human sPLA₂ was purchased from Upstate Biotechnology (Lake Placid, N.Y.) and used as the primary antibody at 0.5 µg/ml. A 1:2,000 dilution of sheep anti-mouse IgG that had been conjugated to horseradish peroxidase (Amersham) was used as the secondary antibody. Light emitted from the luminol substrate after its oxidation by horseradish peroxidase was detected with autoradiography film that was sensitive to blue light (Hyperfilm ECL; Amersham). Densitometry was performed on a Personal Densitometer SI instrument (Molecular Dynamics, Sunnyvale, Calif.) with the manufacturer's ImageQuant software.

Lysozyme. Lysozyme activity was measured by a radial diffusion (lysoplate) assay (30). The assay plates contained 1% agarose and 0.5 mg of lyophilized M. lysodeikticus per ml in 15 ml of 66 mM sodium phosphate buffer (pH 7.0). Serially diluted samples were dissolved in 5 µl of 0.01% acetic acid, placed into 3.2-mm-diameter wells, and incubated overnight at room temperature. Clear zones were measured, and activity was expressed relative to that of highly purified human tear lysozyme standards.

Protein microsequencing. N-terminal sequencing was performed with a Porton model 2090E sequencer (Beckman Instruments, Fullerton, Calif.) after the protein had been reduced, carboxymethylated, and transferred to a polyvinylidene difluoride membrane. Quantitative amino acid analysis was performed by the PicoTag method. The molecular mass of the purified secretory PLA₂ was determined by electrospray ionization-mass spectrometry with a Perkin-Elmer Sciex (Thornhill, Canada) triple-quadrupole electrospray mass spectrometer. The instrument was tuned to resolve the isotopes of the polypropylene glycol-NH₄⁺ (singly charged ion at m/z 906 with 40% valley) and calibrated by flow injection of a mixture of polypropylene glycol 425, 1000, and 2000 in water-methanol (1/1 [vol/vol]) containing 2 mM ammonium formate and 0.1% acetonitrile. Samples were dissolved in water-acetonitrile-formic acid (50/50/0.1) and injected into a 10-µl/min stream of this solvent. Spectra were averaged, and the multiply charged ion series were deconvoluted with the MacSpec and MacBiospec software supplied with the instrument.

Other assays. Protein was measured by the micro-bicinchoninic acid assay, with BSA standards (Pierce, Rockford, Ill.).

	RESULTS

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General composition of tears. As shown in Table 1, basal tears contained 13.4 ± 1.28 mg of protein ml¹ (mean ± standard error) and contained from 8.3 to 17.1 mg of protein ml¹. Onion vapor-stimulated tears contained 8.97 ± 0.93 mg of protein ml¹ and ranged from 5.0 to 11.3 mg of protein ml¹. SDS-PAGE analysis confirmed that lysozyme, lactoferrin, and lipocalin were especially abundant (Fig. 1).

View larger version (81K): [in this window] [in a new window]   FIG. 1.   Purification of secretory PLA2 from tears. The left panel shows an SDS-PAGE gel (16.5% polyacrylamide) run under reducing conditions and stained with Coomassie blue. Its left lane contained molecular mass standards (STD.) with masses of 3.0 (a), 6.2 (b), 14.3 (c), 18.4 (d), 29.0 (e), and 43.0 (f) kDa. Lane A (5 µl of pooled tears) displays three major bands, which have been numbered. They correspond to the following proteins: 1, lysozyme; 2, tear-specific prealbumin (also called lipocalin); and 3, lactoferrin. Lane B shows the PLA2-containing fractions after the first stage of RP-HPLC purification. Lane C shows highly purified PLA2 after the second stage of RP-HPLC purification, with an acetonitrile (ACN) gradient in 0.13% HFBA. The panel on the right shows the sPLA2 peak, monitored at 230 nm, and is from the second HPLC purification step.

Antibacterial activity of tears. We performed colony count experiments to test the antimicrobial activity of whole tears against various gram-positive bacteria, including six strains of S. aureus (two clinical ocular isolates and four MRSA strains), L. monocytogenes, B. subtilis, group B Streptococcus, and a vancomycin-resistant E. faecium strain, 94.132. In these studies, 90 µl of pooled tears and 10 µl (10⁵ CFU) of bacteria were mixed together and incubated for 3 h at 37°C in a shaking water bath. As shown in Table 2, over 99% of the bacteria had been eradicated after 1 h, and essentially complete (99.99%) eradication of the organisms was accomplished by 3 h.

Fractionation of tears. To ascertain which components of human tears were responsible for their activity against gram-positive bacteria, we fractionated normal tears by RP-HPLC. Fractions were collected each minute, lyophilized by vacuum centrifugation, redissolved in acidified water (0.01% acetic acid), and tested against L. monocytogenes EGD in radial diffusion assays. The underlay gels contained 10 mM sodium phosphate buffer, 1% agarose, and 0.3 mg of Trypticase soy broth powder per ml ± 100 mM NaCl, without supplemental calcium. As shown in Fig. 2, two distinct antibacterial peaks were present, one centered around fraction 42 and the other centered around fraction 52. The antimicrobial molecules in fraction 42 were almost equally active under low- and high-salt conditions (10 mM phosphate buffer ± 100 mM NaCl). In contrast, those in fraction 52 were considerably more active under the low-salt conditions (10 mM phosphate buffer). Enzymatic assays revealed that fraction 42 contained the highest PLA₂ activity and that fraction 52 corresponded to the lysozyme peak.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Activity of HPLC fractions. Antimicrobial activity was tested against L. monocytogenes with radial diffusion assays by using low-salt () or high-salt () underlay gels that contained 10 mM sodium phosphate ± 100 mM NaCl. Enzymatic PLA2 activity () was measured with 14C-labeled E. coli. Lysozyme activity () was tested with a lysoplate assay.

Purification of PLA₂. Tears were collected from the six donors, pooled, and subjected to RP-HPLC. When the partially purified fractions with PLA₂ activity were examined by SDS-PAGE, they contained two principal components, both with apparent masses of approximately 14 kDa (Fig. 1, lane B). These proteins were resolved and purified by rechromatography of the fractions on the same C₁₈ column with 0.13% HFBA as the ion-pairing reagent. The partial N-terminal sequence (residues 1 to 22) of the more abundant protein was identical to that of human type II sPLA₂: NLVNF HRMIK LTTGK EAALS YG. The molecular mass of the purified molecule was 13,905.2 Da by electrospray ionization-mass spectrometry which was in close agreement with the expected mass of 13,903.7, calculated from the molecule's primary sequence (36). In addition, the purified molecule reacted with a monoclonal antibody to human sPLA₂ in Western blots (data not shown). This constellation of findings (N-terminal sequence, mass, and immunological reactivity) securely established the molecule's identity as sPLA₂. The N-terminal sequence of the other peptide present in Fig. 1, lane B, corresponded precisely to that of serum leukoprotease inhibitor (SLPI), and this molecule also reacted strongly to a polyclonal antibody against human recombinant SLPI (data not shown). Figure 1, lane C, shows highly purified sPLA₂, after its resolution from SLPI by the second RP-HPLC. The right panel of Fig. 1 shows the RP-HPLC chromatogram of purified human tear sPLA₂.

Quantitation of tear sPLA₂. The sPLA₂ concentration in tears of six healthy individuals was measured by two methods: one enzymatic and the other immunochemical. The enzymatic assay used [¹⁴C]oleate-labeled autoclaved E. coli as a substrate. The immunoassay was an ECL procedure using a murine monoclonal antibody to human sPLA₂. Highly purified human tear PLA₂ was used as the standard in both assays. In Western blotting experiments with whole tears, the monoclonal antibody stained only sPLA2 and detected <1 ng of sPLA₂ when we used an alkaline phosphatase-conjugated secondary antibody (data not shown). The ECL assay was even more sensitive, and its output on film was better suited to densitometry. Figure 3 shows a representative standard curve from the ECL assay of sPLA₂.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   ECL assay. A standard curve is shown for sPLA2 purified from human tears.

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Antimicrobial activity of tear PLA₂. Purified PLA₂ was highly effective against all of the gram-positive bacteria we tested (Fig. 4 and 5). The minimal effective concentration of sPLA₂ against the various organisms, indicated by their respective x-intercepts, varied over a large range in the experiments shown in Fig. 4. Whereas L. monocytogenes EGD was killed by 1.1 ng of sPLA₂ ml¹ (0.08 nM), approximately 250 ng of sPLA₂ ml¹ (18 nM) was needed to kill the least-sensitive organism in this group, E. faecium 94.132. Two S. aureus strains showed intermediate sensitivity, requiring 15 and 80 ng of sPLA₂ ml¹ (1.1 and 5.8 nM, respectively).

View larger version (19K): [in this window] [in a new window]   FIG. 4.   Antibacterial activity of purified sPLA2. Two-stage radial diffusion assays were performed with highly purified tear sPLA2 and five gram-positive bacteria: L. monocytogenes (), S. aureus 67395 (), S. aureus GM-1 (), group B streptococcus (), and E. faecium 94.132 (). Each symbol represents a mean value derived from three separate experiments with each organism. The regression lines were fit by the method of least mean squares. The x-intercepts indicate the minimal effective concentration.

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View larger version (23K): [in this window] [in a new window]   FIG. 5.   Susceptibility of S. epidermidis and M. luteus. Radial diffusion assays were performed with purified human tear sPLA2, human tear lysozyme, and human milk lactoferrin. Note that the MIC (x-intercept) of sPLA2 for M. luteus () was approximately 0.3 µg/ml, whereas that of lysozyme was 13 µg/ml. The MIC of sPLA2 for S. epidermidis () was approximately 0.15 µg/ml, whereas lysozyme was not effective (MIC of >1.5 mg/ml).

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Rapidity of the effect. Figure 6 shows that sPLA₂ acted rapidly after addition to 1.5 × 10⁶ to 3 × 10⁶ bacteria ml¹ in artificial tear fluid. After 15 min of incubation, as little as 100 ng of sPLA₂ ml¹ totally eradicated L. monocytogenes EGD and 5 µg ml¹ eradicated S. aureus 67395. 

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Colony count assay. Approximately 106 CFU per ml of L. monocytogenes (L. mono.) EGD or S. aureus 67395 was incubated for 15 min at 37°C with the indicated concentrations of tear sPLA2.

Effects of divalent cations. To determine the degree to which the bactericidal activity of sPLA₂ was calcium dependent, we performed the experiments shown in Fig. 7. The basic unsupplemented underlay gels contained 1% agarose, full-strength calcium- and magnesium-free ATS, and 0.3 mg of Trypticase soy broth powder per ml. The addition of 0.7 mM CaCl₂ to these underlay gels enhanced the bactericidal potency of PLA₂ over 1,000-fold, whereas addition of 2 mM EGTA, a selective Ca²⁺ chelator, abolished its antimicrobial activity even when the gels also contained 0.7 mM calcium. The slight activity of sPLA₂ in underlay gels without specifically added calcium (Fig. 7, solid circles) probably reflects the effects of the calcium contained in Trypticase soy broth powder, since it was also abolished by addition of EGTA. In retrospect, we were lucky to have selected L. monocytogenes, which is exquisitely sensitive to sPLA₂, for the preliminary studies shown in Fig. 2, since the very low concentrations of calcium that were present in the underlay were markedly suboptimal for sPLA₂.

View larger version (24K): [in this window] [in a new window]   FIG. 7.   Calcium dependence of antibacterial activity. Tear sPLA2 was tested against two gram-positive bacteria, L. monocytogenes and S. aureus. The underlay gels contained ATS without Ca2+ or Mg2+ and were supplemented with divalent cations (0.7 mM) or EGTA (2 mM), as indicated by the inset. Each symbol depicts a mean value derived from four separate experiments.

Antimicrobial activity of other tear components. The calcium dependence of sPLA₂-mediated antimicrobial activity against gram-positive bacteria and especially its abolition by EGTA provided a facile method for determining if additional antimicrobial molecules contributed to the antimicrobial properties of tears against gram-positive bacteria. As shown in Table 3, the addition of 2 mM EGTA to normal tears completely blocked its activity against all six S. aureus strains, including the four MRSA strains. In contrast, the activity of tears against L. monocytogenes and group B Streptococcus was only partially blocked, and EGTA-containing tears retained their activity against E. faecium and B. subtilis.

	DISCUSSION

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Human sPLA₂ is a 13.9-kDa molecule composed of 124 amino acid residues. It has been found in many tissues and secretions, including rheumatoid synovial fluids (37), platelets (21), seminal plasma (29, 41), intestinal Paneth cells (27), and neutrophils (32). The levels of sPLA₂ in seminal plasma were reported to range between 15 and around 30 µg/ml (29)very similar to the levels we found in the tear film (Table 1). It is noteworthy that sPLA₂ hydrolyzes phosphatidylglycerol several hundred times more rapidly than phosphatidylcholine, since the former is a principal phospholipid of microbial membranes, whereas the latter is typically abundant in mammalian cell membranes (24, 25, 44).

All PLA₂ enzymes (EC 3.1.1.4) hydrolyze the sn-2 fatty acyl moiety from phospholipids, releasing equimolar amounts of free fatty acids and lysophospholipids. Over 60 secretory PLA₂ enzymes were reviewed by Scott and Sigler (34, 35), who described sPLA₂ enzymes as robust, small molecules that were highly resistant to denaturation and preferred substrates that were organized into micelles, monolayers, or membranes. All sPLA₂ enzymes bind calcium ions with a K_d of >10⁴ M, and this cation is essential for catalysis. The various PLA₂ enzymes show strongly conserved three-dimensional structures that are stabilized by multiple intramolecular cystine disulfide bonds. Although mammalian sPLA₂s are often described according to their tissues of origin, (e.g., pancreatic, splenic, or intestinal PLA₂), only a single sPLA₂ gene exists in humans (37). Type I and type II PLA₂ enzymes are distinguished by the location of one of their seven disulfide bridges and by a seven-residue C-terminal extension found only in the type II PLA₂s. Type I PLA₂ is exemplified by mammalian pancreatic enzymes and homologs found in Old World elapid snake venoms, whereas the type II enzymes include intestinal and splenic PLA₂s and venom constituents of New World viperid and crotalid snakes (16, 18). Additional PLA₂ enzymes that are structurally and functionally distinct from the type I and II enzymes are also found in mammalian cells and may be especially important in mediating cellular injury (3, 33).

Our experiments demonstrated that remarkably large concentrations of type II sPLA₂ are present in normal human tears. Only one previous report described the presence of sPLA₂ in normal human tears (26). Using a time-resolved fluoroimmunoassay procedure, these investigators reported that tears contained 1.45 µg of sPLA2 ml¹, a value 10- to 20-fold lower than the sPLA₂ concentrations shown in Table 1. Since we used both an enzymatic assay and an immunoassay procedure to determine these sPLA₂ concentrations, we are confident that the concentrations shown in Table 1 are accurate. This belief is reinforced by our ability to recover 10.4 µg of highly purified sPLA₂ ml¹ from tears by the two-stage HPLC procedure described above. The earlier study of sPLA₂ in tears was performed in a country (Finland) whose population is unusually homogeneous (15). Consequently, it is noteworthy that the two ethnic Finns in our donor group had sPLA₂ concentrations in their tears similar to those found in the Asian or Caucasian donors (Table 1).

Whereas we obtained nearly identical values (36.7 versus 32.1 µg/ml) for the concentration of sPLA₂ in basal tears with the enzymatic and immunological assays, the assays gave divergent results (27.4 versus 14.9 µg/ml) when applied to onion vapor-stimulated tears. Our preliminary evidence suggests that the lower sPLA₂ activity in stimulated tears reflects the presence of an sPLA₂ inhibitor, as yet unidentified (data not shown). Endogenous sPLA₂ inhibitors also exist in bovine seminal plasma (22).

Our data provide compelling evidence that sPLA₂ is principally responsible for the ability of tears to kill a broad spectrum of gram-positive bacteria, notwithstanding the presence of lysozyme and lactoferrin in much higher concentrations. This inference is supported by several lines of evidence. First, concentrations of purified human sPLA₂ much lower than those present in tears showed potent bactericidal activity against each of the gram-positive bacteria in our panel. This bactericidal activity was calcium dependent and was also inhibited by EGTA, suggesting that the enzymatic effects of sPLA₂ were critical for its bactericidal properties. Second, 2 mM EGTA abolished the bactericidal effect of normal tears against normal and MRSA strains and greatly reduced their activity against group B streptococci and L. monocytogenes (Tables 2 and 3). Lysozyme and lactoferrin displayed little or no activity against these organisms, even when tested at 1.5 mg/ml. As might be expected, EGTA did not inhibit the activity of tears against highly lysozyme-susceptible bacteria, such as B. subtilis, and a vancomycin-resistant strain of E. faecium.

Not withstanding its high concentration, our data indicate that lysozyme acts in a secondary (backup) manner with respect to the bactericidal properties of human tears against gram-positive bacteria. The reported absence of lysozyme from the tears of cattle is consistent with its secondary role in this respect (31). Inbred mouse strains, including the widely used C57BL/6 strain, that are naturally deficient in sPLA2 because of a frameshift mutation in exon 3 of the gene (19) may afford useful models for defining the role of sPLA₂ in mucosal and secretory host defenses.

Both murine intestinal (14) and rabbit leukocyte (45) sPLA₂ possess bactericidal properties. The potent activity of rabbit leukocyte sPLA₂ against S. aureus largely accounted for the staphylocidal activity of a sterile inflammatory peritoneal exudate fluid which contained 10 nM (0.14 µg/ml) of sPLA₂ (45). Whereas normal human serum contains low levels of sPLA₂ that circulate mostly in high-molecular-weight complexes (28), the concentration of sPLA₂ in human serum rises sharply during sepsis (6, 12). sPLA₂ causes bacterial phospholipid degradation during phagocytosis of E. coli cells by polymorphonuclear leukocytes and also degrades the phospholipids of E. coli cells treated with neutrophil-derived bactericidal, permeability-increasing protein (46).

The remarkable susceptibility of L. monocytogenes to human secretory PLA₂ (Fig. 4) has an ironic aspect, since this organism uses two secreted phospholipasesa phosphatidylinositol-specific phospholipase C and a broad-range phospholipase Cto escape from vacuoles of the host's phagocytes and spread from cell to cell (2, 39).

The development of molecules that can inhibit PLA₂ activity is a major area of pharmaceutical research (11, 42), in part stimulated by the belief that the elevated concentrations of sPLA2 in the inflammatory fluids, plasma, and infected tissues are noxious. The present report and other recent demonstrations (14, 45) that sPLA₂ has potent microbicidal properties suggest that its induction and release may be beneficial to hosts with infections caused by gram-positive bacteria. Should sPLA₂ inhibitors enter into routine clinical use, it will be important to be watchful for evidence of impaired host resistance or of increased infections caused by gram-positive organisms.