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(Investigative Ophthalmology and Visual Science. 1999;40:2744-2748.)
© 1999 by The Association for Research in Vision and Ophthalmology, Inc.

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

Cheol Hwa Song1, Jae Sin Choi2, Dae Kyong Kim2 and Jae Chan Kim1

1 From the Department of Ophthalmology, Chung–Ang University Hospital and 2 Department of Environmental and Health Chemistry, College of Pharmacy, Chung–Ang University, Seoul, Korea.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
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 McCulley1 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
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
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.


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  		Table 1. PLA2 Activity in Each Group of Chronic Blepharitis Patients and in Normal Controls

 
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 800 µl supernatant was counted for radioactivity in a liquid ß-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.



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  		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.

 
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
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
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) .



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  		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).

 


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  		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.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
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.


   Footnotes
 
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.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Dougherty, JM, McCulley, JP (1984) Comparative bacteriology of chronic blepharitis Invest Ophthalmol Vis Sci 68,524-528
  2. Holly, FJ (1985) Physical chemistry of the normal and disordered tear film Trans Ophthalmol Soc UK 104,374-380
  3. Nicolades, N, Kaitaranta, JK, Rowdah, TN, Macy, JI, Boswell, FM, Smith, RE (1981) Meibomian gland studies: comparison of Steer and human lipids Invest Ophthalmol Vis Sci 20,522-528[Abstract/Free Full Text]
  4. Greiner, JV, Glonek, T, Korb, DR, Leahy, CD (1996) Meibomian gland phospholipids Curr Eye Res 15,371-375[Medline][Order article via Infotrieve]
  5. Shine, WE, McCulley, JP (1991) The role of cholesterol in chronic blepharitis Invest Ophthalmol Vis Sci 32,2272-2280[Abstract/Free Full Text]
  6. Bonventre, JV. (1992) Phospholipase A2 and signal transduction J Am Soc Nephrol 3,128-150[Abstract]
  7. Gronich, JH, Bonventre, JV, Nemenoff, RA (1988) Identification and characterization of a hormonally regulated form of phospholipase A2 in rat renal mesangial cells J Biol Chem 263,16645-16651[Abstract/Free Full Text]
  8. Thakur, A, Willcox, MDP, Stapleton, F. (1998) The proinflammatory cytokines and arachidonic acid metabolites in human overnight tears: homeostatic mechanisms J Clin Immunol 18,61-70[Medline][Order article via Infotrieve]
  9. Kim, DK, Fukuda, T, Thompson, BT, Cockrill, B, Hales, C, Bonventre, JV (1995) Bronchoalveolar lavage fluid phospholipase A2 activities are increased in human adult respiratory distress syndrome Am J Physiol 269,L109-L118[Abstract/Free Full Text]
  10. Dole, VP, Meinertz, H. (1960) Microdetermination of long-chain fatty acids in plasma and tissues J Biol Chem 235,2595-2599[Free Full Text]
  11. Bradford, MM (1976) A rapid and sensitive method for the quantitation of microorganism quantities of protein utilizing the principle of protein dye binding Anal Biochem 72,248-254[Medline][Order article via Infotrieve]
  12. Bazan, HEP (1987) Corneal injury alters eicosanoid formation in the rabbit anterior segment in vivo Invest Ophthalmol Vis Sci 28,314-319[Abstract/Free Full Text]
  13. Helleboid, L, Khatami, M, Wei, ZG, Rockey, JH (1991) Histamine and prostacyclin. Primary and secondary release in allergic conjunctivitis Invest Ophthalmol Vis Sci 32,2281-2289[Abstract/Free Full Text]
  14. Gronich, JH, Bonventre, JV, Nemenoff, RA (1988) Identification and characterization of a hormonally regulated form of phospholipase A2 in rat renal mesangial cells J Biol Chem 263,16645-16651
  15. Vadas, P, Pruzanski, W. (1986) Biology of disease: role of secretory phospholipase A2 in the pathobiology of disease Lab Invest 55,391-404[Medline][Order article via Infotrieve]
  16. Aho, HJ, Saari, K, Kallajoki, M, Nevalainen, TJ (1996) Synthesis of group II phospholipase A2 and lysozyme in lacrimal gland Invest Ophthalmol Vis Sci 37,1826-1832[Abstract/Free Full Text]
  17. Bazan, HE, Varner, L. (1997) A mitogen-activated protein kinase (MAP-kinase) cascade is stimulated by platelet activating factor (PAF) in corneal epithelium Curr Eye Res 16,372-379[Medline][Order article via Infotrieve]
  18. Bazan, NG, Allan, G. (1997) Signal transduction and gene expression in the eye: a contemporary view of the pro-inflammatory, anti-inflammatory and modulatory roles of prostaglandins and other bioactive lipids Surv Ophthalmol 41,s23-s34



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