Enhanced Secretory Group II PLA₂ Activity in the Tears of Chronic Blepharitis Patients

Abstract

purpose. Phospholipase A₂ (PLA₂) hydrolyzes phospholipids, one of the important constituents of human meibomian gland secretions. This study was performed to investigate PLA₂ type and activity in the tears of chronic blepharitis patients compared to those of normal persons.

methods. Tear samples of 36 patients and 10 normal persons were collected in non-heparinized microcapillary tubes. PLA₂ activity in the tears was measured by Dole’s method, and the results of the blepharitis patients were compared to those of the normal persons. The characterization of PLA₂ was performed by the head group preference test and the dithiothreitol (DTT) sensitivity test. The classification of PLA₂ type was done using Western blot analysis with anti-human secretory PLA₂ antibody.

results. No statistically significant differences were found among the six categories of chronic blepharitis. However, the mean PLA₂ activity in the tears of the chronic blepharitis patients was about two times higher than that of the normal controls with statistical significance (P < 0.05). The PLA₂ substrate specificity test revealed group II PLA₂ activity. Furthermore, the group II PLA₂ was identified as a 14 kDa band in Western blot analysis using an antibody raised against human secretory group II PLA₂.

conclusions. Secretory group II PLA₂ activity was significantly enhanced in the tears of the chronic blepharitis patients compared with that of the normal controls. It is suggested that this increased enzymatic activity may decrease the tear film stability through increased hydrolysis of phospholipids.

Chronic blepharitis, a commonly encountered condition, is characterized by variable pictures of eyelid inflammation and is difficult to manage. 

There are several classifications of chronic blepharitis. Dougherty and McCulley ¹ have expanded the two fundamental categories of marginal lid inflammation into six categories, while at the same time placing a strong emphasis on the role of the meibomian glands in the inflammatory picture. 

The tear film lipid layer, derived primarily from secretions of meibomian glands, is important in tear film stability. ² The composition of human meibomian gland secretions have been reported as hydrocarbons (7%), sterol esters (27%), wax esters (32%), triglycerides (4%), polar lipids (15%), free sterols (2%), and free fatty acids (2%). ³ Significant differences have been discovered in meibomian lipid composition between chronic blepharitis patients and normal individuals. The constituents of polar lipids as well as sterol esters and wax esters are important to the tear film stability. ^{4 5} Greiner et al. ⁴ have reported the characteristics of the polar lipids of meibomian gland, especially two major phospholipids (PL), i.e., phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which comprise nearly 60% of the total phospholipid profile and serve as the substrates for a variety of phospholipases. 

It has been known that mammalian cells contain several forms of PLA₂, which can be classified into secretory and cytosolic forms on the basis of their biochemical properties, localization, and primary structures. ⁶ Although it has been suggested that the 14-kDa secretory form of PLA₂ may be involved in inflammatory responses, the 100-kDa cytosolic PLA₂ is thought to be implicated in signal transduction of many cell types. ^{6 7} Whereas secretory forms of PLA₂, which requires millimolar concentrations of Ca²⁺ ions for the activity, exhibit essentially no acyl-chain selectivity, prefer PE to PC, and are sensitive to pretreatment with dithiothreitol (DTT), cytosolic PLA₂ has a high selectivity for phospholipids with an sn-2 arachidonyl chain, hydrolyzes both PE and PC effectively, and is not affected by DTT. 

Group II phospholipase A₂s (PLA₂s) are a family of enzymes that hydrolyze the fatty acid ester bond at the sn-2 position of membrane phospholipids. The released arachidonic acid (AA) is then converted to inflammatory mediators, such as prostaglandins and leukotrienes. In the recent study, leukotriene B₄ and platelet-activating factor have been reported as constituents of the tears. ⁸ These mediators can be involved in the ocular surface inflammations, which means that PLA₂ may be associated in upstream pathway of these mediators. 

In the present study, we examined which type of PLA₂ is involved in chronic blepharitis, an external ocular surface inflammation, and its activity in the tears of these patients compared with that of normal individuals. 

Patients and Methods

Selection of Patients

This research followed the tenets of the Declaration of Helsinki. Informed consent was obtained from the subjects after explanation of the study, and approval was obtained from the appropriate institutional boards. Inclusion criteria were the presence of one or more of the signs and symptoms of blepharitis for more than 6 months without any therapy for at least the preceding 2 weeks. The patients were classified as previously described (Table 1) . ¹ In brief, the six categories of blepharitis were classified as follows: (1) staphylococcal (STAPH); (2) seborrheic, alone (SBBL); (3) seborrheic with a clinical appearance of staphylococcal infection (MIX); (4) seborrheic with meibomian seborrhea (MBSB); (5) seborrheic with spotty inflammation of the meibomian glands (secondary meibomitis, 2MEIB); and (6) severe inflammation of the meibomian glands (meibomian keratoconjunctivitis [MKC]). Normal persons, matched for age and sex to the patients, were selected as controls if they were free of any sign or symptom of ocular disease. 

Based on clinical findings, as described in previous works, ¹ each group of blepharitis patients consisted of six patients, and the normal control group consisted of 10 persons (Table 1) . The subjects were aged between 30 and 40 years. 

Materials

1-Acyl-2-[1-¹⁴C]arachidonyl-sn-glycerol-3-phosphoethanolamine (2-[1-¹⁴C]AA-GPE; 55 mCi/mmol) and 1-stearoyl-2-[1-¹⁴C]arachidonyl-sn-glycerol-3-phosphocholine (2-[1-¹⁴C]AA-GPC; 55.6 mCi/mmol) were purchased from the radio-chemical center (Amersham, Buckinghamshire, UK). A DEAE-5PW column was purchased from the Tosoh Co. (Tokyo, Japan). Nonheparinized microhematocrit tubes (Chase Co., GA) were used for collection of the tears. Group I PLA₂ and group II PLA₂ were purchased from Sigma Co. (St. Louis, MO). Mouse anti-human secretory group II PLA₂ antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). All other chemicals were of highest purity available from commercial sources. 

PLA₂ Assay

The tear samples of the above patients and 10 normal persons were obtained in microhematocrit tubes from the lateral portion of the inferior fornix area without irritation of the conjunctiva between 10 AM and noon. The PLA₂ activity in the tears was assayed by measuring the release of [1-¹⁴C]AA from 1-acyl-2-[1-¹⁴C]arachidonyl-sn-glycerol-3-phosphethanolamine (2-[1-¹⁴C]AA-GPE). The assay was performed as described by Kim et al. ⁹ It was initiated by the addition of 70 μg protein into each tear samples, ^{9 10} with the protein concentration of the tears having been determined with Bradford reagent (Bio-Rad Laboratories, Melville, NY) using bovine serum albumin as a standard. ¹¹ In brief, 2-[1-¹⁴C]AA-GPE was dried under a nitrogen stream and resuspended in absolute ethanol. The standard incubation system (100 μl) for the assay of PLA₂ activity contained 75 mM Tris/HCl (pH 7.5), 5 mM CaCl₂, and 5 μM radioactive phospholipids. The reaction was carried out at 37°C for 30 minutes and was stopped by adding 0.56 ml of Dole’s reagent (n-heptane/isopropylalcohol/1N H₂SO₄; 400:390:10, by volume). The [1-¹⁴C]AA released was extracted as follows: Water (110 μl) was added, and the sample was vortex-mixed and centrifuged at 10,000g for 2 minutes. Then 150 μl of the upper phase was transferred to a new tube, to which 25 mg silica gel and 800 μl n-heptane were added. The samples were vortex-mixed and centrifuged again for 2 minutes, after which 800μ l supernatant was counted for radioactivity in a liquidβ -scintillation counter. 

The PLA₂ activity in each group of the patients was compared with that in the normal group using analysis of variance and the Mann–Whitney test. 

Characterization of PLA₂ Activity in the Tears

T examine which type of PLA₂ exists in the tears, the preference of PLA₂ for the head group of the substrate and the sensitivity of the enzyme to DTT were performed as described previously. ¹² Briefly, each tear sample (1.2 mg protein) was applied to a DEAE-5PW high-performance liquid chromatography column preequilibrated with a buffer containing 50 mM Tris/HCl (pH 7.5) and 1 mM EDTA. Proteins bound to the column were eluted with a 20-ml linear gradient of 0.0 to 0.5 M NaCl. Each fraction was collected and assayed for its PLA₂ activity using 2-[1-¹⁴C]AA-GPC and 2-[1-¹⁴C]AA-GPE as substrates, respectively. Whereas group II PLA₂ revealed higher activity with 2-[1-¹⁴C]AA-GPE than with 2-[1-¹⁴C]AA-GPC (Fig. 2) , group I PLA₂ hydrolyzed both 2-[1-¹⁴C]AA-GPE and 2-[1-¹⁴C]AA-GPC effectively. 

DTT sensitivity testing was performed to differentiate group I or II from cytosolic PLA₂. It is known that DTT abolishes group I or II PLA₂ activity, but does not affect the cytosolic PLA₂ activity. The active PLA₂ fractions eluted from the DEAE-5PW column were preincubated with 2 mM DTT for 10 minutes at 37°C, followed by addition of the substrate, 2-[1-¹⁴C]AA-GPE, and this result was compared with the PLA₂ activity without DTT. These two tests (with DTT and without DTT) were performed with the tears of blepharitis patients and the purified control enzymes, group I, group II, and cytosolic PLA₂. 

Immunoblotting Analysis

Proteins in the patients’ tears were separated by SDS-PAGE (15% polyacrylamide gel) and then electrophoretically transferred to nitrocellulose membranes in 25 mM Tris-HCl (pH 8.3), 190 mM glycine, 20% methanol. Nonspecific binding of anti-human secretory group II PLA₂ antibody to nitrocellulose was prevented by preincubation of the nitrocellulose in 5% skim milk in Tris-buffered saline (TBS; 25 mM Tris-HCl, pH 8.0, 137 mM KCl) for 2 hours at room temperature. The blocked nitrocellulose membrane was incubated with diluted antiserum for more than 6 hours at room temperature with constant shaking. Unbound antibodies were removed with three washes of TBS containing 0.1% Tween 20, and the site of antibody binding was detected by using alkaline phosphatase-conjugated secondary antibody and a NBT-BCIP Substrate Kit (Pierce Co., Rockford, IL). 

Results

The mean PLA₂ activity in the tears of the chronic blepharitis patients was about two times higher than in the normal persons, which was statistically significant (P < 0.001) (Table 1) . However, there were no statistically significant differences in mean PLA₂ activities among each group of blepharitis patients (Table 1) . 

To examine which type of PLA₂ activity was enhanced in the tears of chronic blepharitis patients, the PLA₂ substrate specificity test was performed. A feature characteristic of group II PLA₂ is that it preferentially hydrolyzes 2-[1-¹⁴C]AA-GPE to 2-[1-¹⁴C]AA-GPC by a ratio of about 3:1, whereas the group I PLA₂ hydrolyzes both 2-[1-¹⁴C]AA-GPE and 2-[1-¹⁴C]AA-GPC essentially equally (Fig. 1) . In the DTT sensitivity test (Fig. 2) , PLA₂ activity was inhibited by pretreatment with 5 mM DTT. Under the same conditions that this reducing agent did markedly reduce group I or II PLA₂ activity, it did not affect the cytosolic PLA₂ activity. Thus, these results suggest that the PLA₂ identified in the human tears may be categorized as a form of group II PLA₂. This finding is further supported by the result obtained from the western blot analysis using an anti-human secretory group II PLA₂ antibody, where the PLA₂ protein of the human tears migrated as a molecular mass of 14 kDa, identical with that of the rat platelet group II PLA₂ (Fig. 3) . 

Discussion

Phospholipid, one of the components of the meibomian gland secretions, is believed to serve as the surfactant that forms the interface between the aqueous layer of the tear film and the hydrophobic non–polar lipids comprising the bulk of the oil layer of the tear film. ⁴ Phospholipid plays an important role in the spreading of this non–polar ester and hydrocarbon component over the aqueous component covering the ocular surface. Therefore, it is conceivable that the disruption of phospholipid will cause breakdown of the tear film and alteration of its lipid composition, ^{4 5} and thus produce the signs and symptoms associated with chronic blepharitis. At the present time, we suggest that the disruption of PL, involving the disease, may be caused by the activation of PLA₂. 

On the other hand, although it remains to be clarified, it also is possible that the increase in PLA₂ activity may contribute to inflammation through the production of eicosanoids in the tears. In this context, it was recently reported that a high level of prostaglandins was identified in the tears of patients with allergic conjunctivitis. ¹³ Thus, our results further support the hypothesis that an inflammatory process such as the chronic blepharitis may be attributable to a high level of PLA₂ activity in the tears of these patients. 

We propose two mechanisms for the enhancement of secretory group II PLA₂ activity in the tears of chronic blepharitis patients. First, lipopolysaccharides and endotoxins of bacteria activate inflammatory cells, such as polymorphonuclear leukocytes and macrophages. The activated inflammatory cells appear to release a number of inflammatory mediators, such as eicosanoids, through the induction and/or secretion of secretory group II PLA₂ and thus lead to aggravation of the inflammatory process. Previous works have shown that secretory group II PLA₂ is secreted extracellularly from a number of inflammatory cells when exposed to a diverse spectrum of stimuli, such as calcium ionophore and zymosan. ¹⁴ It is also known that activation of PLA₂ causes the release of other inflammatory mediators, such as histamine and serotonin from the activated basophils, mast cells, and platelets. ¹⁵ Second, the increase in secretory group II PLA₂ activity in the tears is known to be due to its secretion from lacrimal glands. ¹⁶ Finally, recently it was suggested that PLA₂ activity in the eye tissues could be enhanced in the external ocular surface, including the corneal epithelium, in response to injury. ^{17 18}  

In summary, PLA₂ activity was detected in the tears of chronic blepharitis patients and the mean activity of the enzyme in these patients was higher than in the normal controls. The enhanced PLA₂ activity was identified to be due to secretory group II PLA₂ by biochemical characterization of the enzyme and immunochemical study. This study suggests a role for secretory group II PLA₂ in the pathogenesis of chronic blepharitis. 

 Supported in part by the Chung–Ang University Special Research Grants in 1997 (DKK, JCK).

 Submitted for publication October 16, 1998; revised May 18, 1999; accepted June 3, 1999.

 Commercial relationships policy: N.

 Corresponding author: Jae Chan Kim, Chung–Ang University Hospital, Hangang–Ro 3ka 65-207, Yongsan 140-757, Seoul, Korea.

 

Table 1.

 

PLA₂ Activity in Each Group of Chronic Blepharitis Patients and in Normal Controls

Groups		PLA2 activity (cpm/protein unit × 103)*	Mean ± SD (cpm/protein unit × 103)
STAPH	(n = 6)	11.71 ± 0.59
SBBL	(n = 6)	11.67 ± 0.69
MIX	(n = 6)	14.01 ± 1.12	13.17 ± 1.76, †
MBSB	(n = 6)	12.38 ± 1.11
2MEIB	(n = 6)	13.13 ± 0.90
MKC	(n = 6)	16.12 ± 0.42
Normal	(n = 10)	7.77 ± 0.92	7.77 ± 0.92, †

 STAPH, staphylococcal; SBBL, seborrheic alone; MIX, seborrheic with a staphylococcal infection; MBSB, seborrheic with meibomian seborrhea; 2MEIB, secondary meibomitis (seborrheic with spotty inflammation of the meibomian glands); MKC, meibomian keratoconjunctivitis (severe inflammation of the meibomian glands).

*  Values are means ± SD.

†   P < 0.001.

Figure 1.

 

The substrate specificity test in tears of chronic blepharitis (B). The PLA₂ in tears of chronic blepharitis preferentially hydrolyzed 2-[1-¹⁴C]AA-GPE to 2-[1-¹⁴C]AA-GPC by a ratio of about 3:1, a characteristic feature of group II PLA₂ (C), whereas the Group I PLA₂ hydrolyzed both 2-[1-¹⁴C]AA-GPE and 2-[1-¹⁴C]AA-GPC essentially equally (A).

Figure 2.

 

In the dithiothreitol (DTT) sensitivity test, the PLA₂ activity of tears in chronic blepharitis was inhibited markedly by DTT, a characteristic feature of group I or II PLA₂. DTT markedly reduced group II PLA₂ activity, but did not affect the cytosolic PLA₂.

Figure 3.

 

Western blot analysis of human tears using anti-human secretory group II PLA₂ antibody. Fourteen-kilodalton bands, the range of group II PLA₂ molecular weight, were remarkable in the tears of chronic blepharitis patients (BL) compared to normal controls (NL). M, molecular marker.