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(Investigative Ophthalmology and Visual Science. 2001;42:318-320.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Group II PLA2 Content of Tears in Normal Subjects

K. Matti Saari1, Valtteri V. Aho1, Ville Paavilainen1 and Timo J. Nevalainen2

1 From the Departments of Ophthalmology and 2 Pathology, University of Turku, Turku, Finland.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the concentration of group II phospholipase (PL) A2, an antimicrobial molecule, in tears of normal subjects in different age and sex groups.

METHODS. PLA2 content of tears was measured in 122 healthy volunteers with ages ranging from 20 to 89 years (mean, 49.5 years) by a time-resolved fluoroimmunoassay using a polyclonal rabbit antibody to recombinant human PLA2.

RESULTS. The mean concentration of PLA2 in tears was 54.5 ± 33.9 µg/ml. It was highest in the age group 20 to 29 years (81.6 ± 32.0 µg/ml), and a decrease of concentration occurred with an increase of age. PLA2 values were statistically significantly lower in the age group 60 to 69 years (P = 0.0013) and 70 years or more (P = 0.0001) than in the age group 20 to 29 years. There were no statistically significant differences in PLA2 content of tears between the genders in any age group (P = 0.798).

CONCLUSIONS. The results indicate that tears contain a high concentration of PLA2 and that PLA2 levels decrease with an increase of age and/or reflex tear component of the sample analyzed.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Tears play an important role in the nonspecific defense against bacterial and fungal invasion by washing away the microbes from the surface of the eye.1 Tears contain several proteins with antimicrobial properties.1 2 3 Alexander Fleming reported the presence of lysozyme in tears and described its ability to lyse leisodeikticus.4 5 Lysozyme is a bacteriolytic enzyme that hydrolyzes ß 1–4 linkages between N-acetylmuramic acid and N-acetylglucosamine that stabilize bacterial peptidoglycans, especially in Gram-positive bacteria.6 The mean lysozyme content of tears in normal subjects is 1700 µg/ml.7 Besides lysozyme, the antimicrobial properties of tears have been explained by their high concentration of lactoferrin, betalysin, complement, and secretory immunoglobulin A.1 3 8

Nevalainen et al.9 were the first to report the presence of phospholipase (PL) A2 in human main lacrimal gland and tears. We observed two specialized cell types in the main and accessory lacrimal glands, one synthesizing group II PLA2 and the other synthesizing lysozyme.10 Lysozyme was present in the secretory granules of most acini, whereas PLA2 was seen in a minority of acinar cells, primarily in the central parts of lobules in the main and accessory lacrimal glands.10

PLA2 is a lipolytic enzyme that catalyzes the hydrolysis of the acyl ester bond at the sn-2 position of phosphoglyserides.11 In humans, several enzyme proteins have been identified, including an 85,000 mol wt intracellular PLA2 and two 14,000 mol wt secretory PLA2s.12 13 14 The secretory PLA2s are divided into two subgroups on the basis of the amino acid sequence.15 Group I PLA2 is present in the pancreas, and group II PLA2 in platelets and different fluids and tissues, for example, in Paneth cells, chondrocytes, and synovial fluid and in prostatic gland cells and seminal plasma.16 17 In tears, group II PLA2 is principally responsible for killing a broad spectrum of Gram-positive bacteria.18

There are no data available on the effect of age and sex on the concentration of PLA2 in tears. In this study, we measured the PLA2 levels in tears of normal subjects in different age and sex groups and show that the content of PLA2 was highest in the age group 20 to 29 years and that a decrease of the PLA2 concentration occurred with an increase in age and/or with a reflex component of the sample.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Subjects
The concentration of PLA2 in tears was studied in 122 healthy volunteers, 60 men and 62 women with ages ranging from 20 to 89 years (mean, 49.5 years; Table 1 ). None of the subjects wore contact lenses. The values for PLA2 were assessed in tear samples from one eye of each healthy subject. The principles of the World Medical Association Declaration of Helsinki were followed. Informed consent was obtained from each subject giving the tear specimen. The study protocol was approved by the Ethics Committee of the University of Turku and the Turku University Hospital.


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  		Table 1. Distribution of Number of Normal Subjects According to Age and Sex

 
Collection of Tears
Nonstimulated tears were collected using disposable 5-µl microcapillaries (Microcaps 5 µl; Drummond Scientific Co., Broomall, PA) or 5- or 10-µl microcapillaries (Duran Ringcaps; Hirschmann Laborgeräte, Eberstadt, Germany) under a Haag-Streit 900 biomicroscope. The samples were gathered from the marginal tear strip of the lower lid near the lateral canthus, with care being taken not to irritate the conjunctiva, cornea, or lid margin. Usually (97 subjects), a 5-µl tear sample was taken using a microcapillary, but if the tear production was exceptionally scarce (1.0–4.9 µl in 15 subjects) or plentiful (5.1–7.0 µl in 8 subjects and 20 µl in 2 subjects), an additional calibrating scale was used beside the microcapillary. In all cases the collection time was limited to 5 minutes. The tear samples were diluted from 1:300 to 1:1100 with physiological saline and kept frozen at -70°C until assayed.

PLA2 Assay
The concentration of PLA2 in tear fluid was measured by a time-resolved fluoroimmunoassay using a polyclonal rabbit antibody to recombinant human group II PLA2.19 The results are expressed as micrograms per milliliter (µg/ml).

Statistical Analysis
PLA2 values in different age and sex groups were compared using a two-way analysis of variance. After overall test, all six age groups were compared pairwise. The comparisons were made within sex groups as well as for all subjects. Because several comparisons were done simultaneously, Tukey–Kramer multiple comparison method was used to adjust the P values. All P values lower than 0.05 were considered statistically significant. The results are expressed as means ± SD.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The mean concentration of PLA2 in tears of normal subjects was 54.5 ± 33.9 µg/ml in the total material. Analysis using the General Linear Models Procedure showed that the PLA2 values in different age groups were not similar (P = 0.0023). However, there were no differences in the PLA2 values between the genders in different age groups and in the total material (P = 0.798).

The PLA2 content of tears in normal subjects was highest in the age group 20 to 29 years both in men (79.6 ± 29.6 µg/ml), in women (83.7 ± 35.8 µg/ml), and in all subjects (81.6 ± 32.0 µg/ml). A decrease in the concentration of PLA2 occurred with an increase of age from 30 to 70 years or more (Fig. 1) . When compared with the PLA2 values of tears in the age group 20 to 29 years, the PLA2 values of tears were statistically significantly lower in men in the age group 70 years or more (P = 0.046), in women in the age groups 60 to 69 years (P = 0.028) and 70 years or more (P = 0.013), and in the total material in the age groups 60 to 69 years (P = 0.0013) and 70 years or more (P = 0.0001; Table 2 ).



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  		Figure 1. PLA2 concentration (mean ± SD) of tears in different age groups.

 

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  		Table 2. Mean Group II PLA2 Content in Tear Fluid of Healthy Subjects in Different Age Groups

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The results of this study showed that in normal human tears the mean concentration of PLA2 was 54.5 ± 33.9 µg/ml, which is consistent with the normal values (36.7 ± 2.99 µg/ml, mean ± SEM) reported by Qu and Lehrer.18 It is important to notice that the concentration of PLA2 in tears (54.5 µg/ml) exceeded the levels found in normal serum (3.7 µg/l19 ) by four orders of magnitude and also markedly exceeded the concentration of PLA2 in the seminal plasma (13.4 ± 12.7 µg/ml20 ). The present results showed that there were no significant differences in the PLA2 content of tears between the genders as similarly reported earlier on lysozyme secretion.7

The present results showed that the PLA2 content of tears was highest in the age group of 20 to 29 years and that decreasing values with an increase of age from 30 to 70 years or more were seen both in men, women and in the total material. Similarly, the concentration of lysozyme in tears has been shown to be highest in the age group 21 to 40 years, and a decrease of lysozyme concentration occurred with an increase in age from 30 to 40 years.7 Thus, both the PLA2 and lysozyme contents of tears decreased with an increase of age.

In this study the flow rate of tears varied markedly between the subjects, and the volume of tears collected varied between 1 and 20 µl. In this connection, the decrease in the PLA2 content of tears could also be explained by a decrease in reflex tearing with age. However, we found no statistically significant differences in the PLA2 content of nonstimulated tears in subjects with slow secretion compared with subjects with fast secretion (P = 0.82).

PLA2 is principally responsible for the ability of tears to kill a broad spectrum of Gram-positive bacteria. Only 1.1 ng/ml of PLA2 sufficed to kill Listeria monocytogenes and 15 to 80 ng/ml of PLA2 killed Staphylococcus aureus.18 Micrococcus luteus strain was killed by 13 µg/ml of lysozyme and by 0.3 µg/ml of PLA2.18 These concentrations were 130-fold lower than the concentration of 1768 µg/ml of lysozyme in tears7 and 180-fold lower than the concentrations of PLA2 in normal tears (Table 2) . The present results (Table 2) show that even for the older subjects (70 years or older) the levels of PLA2 in tears are considerably above the level required for effective killing of Gram-positive bacteria.

In summary, we found that the mean PLA2 content of tears was 54.5 ± 33.9 µg/ml. It was highest in the age group of 20 to 29 years, and a decrease of PLA2 concentration occurred with an increase in age from 30 to 70 years or more. These findings are consistent with previous evidence that PLA2 plays a substantial antibacterial role in tears.


   Acknowledgements
 
The authors thank Heikki Peuravuori for his help and advice in analytical methods and Virpi Ahola for her help in statistical analysis.


   Footnotes
 
Supported by Turku University Foundation and Turku University Hospital Research Fund.

Submitted for publication September 15, 1999; revised February 28, June 20, and September 13, 2000; accepted October 6, 2000.

Commercial relationships policy: N.

Corresponding author: K. Matti Saari, Department of Ophthalmology, University of Turku, FIN-20520, Turku, Finland. matti.saari{at}tyks.fi


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Mondido, BJ (1991) Host defense against bacterial and fungal disease Tasman, W Jaeger, EA eds. Duane’s Foundations of Clinical Ophthalmology 2,1-11 JB Lippincott Philadelphia.
  2. Saari, KM, Aine, E, Parviainen, MT (1985) Determination of protein content in ocular fluids by high-performance gel filtration chromatography O’Connor, GR Chandler, JW eds. Advances in Immunology and Immunopathology of the Eye ,258-262 Masson New York.
  3. Records, RE (1991) The tear film Tasman, W Jaeger, EA eds. Duane’s Foundations of Clinical Ophthalmology 2,1-22 JB Lippincott Philadelphia.
  4. Fleming, A. (1922) On a remarkable bacteriolytic element found in tissues and secretions Proc R Soc Ser B 93,306-317
  5. Fleming, A, Allison, VD (1922) Observations on a bacteriolytic substance ("lysozyme") found in secretions and tissues Br J Exp Pathol 3,252-260
  6. Van Haeringen, NJ (1981) Clinical biochemistry of tears Surv Ophthalmol 26,84-96[Medline][Order article via Infotrieve]
  7. Saari, KM, Aine, E, Posz, A, Klockars, M. (1983) Lysozyme content of tears in normal subjects and in patients with external eye infections Graefes Arch Clin Exp Ophthalmol 221,86-88[Medline][Order article via Infotrieve]
  8. Kijlstra, A, Kuizenga, A. (1994) Analysis and function of the human tear proteins Adv Exp Biol Med 350,299-308[Medline][Order article via Infotrieve]
  9. Nevalainen, TJ, Aho, HJ, Peuravuori, H. (1994) Secretion of group 2 phospholipase A2 by lacrimal glands Invest Ophthalmol Vis Sci 32,417-421
  10. Aho, HJ, Saari, KM, Kallajoki, M, Nevalainen, TJ (1996) Synthesis of group II phospholipase A2 and lysozyme in lacrimal glands Invest Ophthalmol Vis Sci 37,1826-1832[Abstract/Free Full Text]
  11. van den Bosch, H. (1980) Intracellular phospholipases A Biochim Biophys Acta 604,191-246[Medline][Order article via Infotrieve]
  12. Clark, JD, Lin, L-L, Kriz, RW, et al (1991) A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP Cell 65,1043-1051[Medline][Order article via Infotrieve]
  13. Kramer, RM, Hession, C, Johansen, B, et al (1989) Structure and properties of a human non-pancreatic phospholipase A2 J Biol Chem 264,5768-5775[Abstract/Free Full Text]
  14. Seilhamer, JJ, Pruzanski, W, Vadas, P, Plant, S, Miller, JA, Kloss, J, Johnson, LK (1989) Cloning and recombinant expression of phospholipase A2 present in rheumatoid arthritic synovial fluid J Biol Chem 264,5335-5338[Abstract/Free Full Text]
  15. Heinrikson, RL, Krueger, ET, Keim, PS (1977) Amino acid sequence of phospholipase A2-{alpha} from the venom of Crotalus adamanteus: a new classification of phospholipases A2 based upon structural determinants J Biol Chem 252,4913-4921[Abstract/Free Full Text]
  16. van den Bosch, H, Aarsman, AJ, van Schaik, RHN, Schalkwijk, CG, Neijs, FW, Sturk, A. (1990) Structural and enzymological properties of cellular phospholipases A2 Biochem Soc Trans 18,781-785[Medline][Order article via Infotrieve]
  17. Nevalainen, TJ, Haapanen, TJ (1993) Distribution of pancreatic (group I) and synovial-type (group II) phospholipases A2 in human tissues Inflammation 17,453-464[Medline][Order article via Infotrieve]
  18. Qu, X-D, Lehrer, RJ (1998) Secretory phospholipase A2 is the principal bactericide for staphylococci and other gram-positive bacteria in human tears Infect Immun 66,2791-2797[Abstract/Free Full Text]
  19. Nevalainen, TJ, Kortesuo, PT, Rintala, E, Märki, F. (1992) Immunochemical detection of group 1 and group 2 phospholipases A2 in human serum Clin Chem 38,1824-1829[Abstract/Free Full Text]
  20. Nevalainen, TJ, Meri, K-M, Niemi, M. (1993) Synovial-type (group II) phospholipase A2 in human seminal plasma Andrologia 25,355-358[Medline][Order article via Infotrieve]



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