Non-Lysozyme Antibacterial Factor in Human Tears

NON-LYSOZYME ANTIBACTERIAL FACTOR IN HUMAN TEARS BETH R. FRIEDLAND,* DOUGLAS R. ANDERSON, M.D.,

AND RICHARD K. FORSTER,

M.D.

Miami, Florida Lysozyme is known to be present in rela­ tively high concentrations in tears. It has been generally assumed, as first suggested by Fleming,1 that lysozyme serves the function of destroying harmful bacteria in the tear film. However, in 1941, Thompson and Gallardo2 showed that lysozyme was not the only antibacterial substance present in tears, and that another heat-sensitive substance was the primary factor responsible for the antistaphylococcal activity of tears. In addi­ tion, it is now well accepted that the antibac­ terial activity of lysozyme is limited to a few Gram-positive bacteria that contain in their cell walls certain mucopolysaccharides that are sensitive to lysis by lysozyme.3 The present study was undertaken to con­ firm the presence of this non-lysozyme anti­ bacterial factor ( N L A F ) and to study some of its characteristics. MATERIALS AND METHODS

Tears were obtained from eight healthy volunteers (ages IS to 35 years) from the staff of the Bascom Palmer Eye Institute. For the lysoplate determinations, tears were collected on disks of filter paper 7 mm in di­ ameter by holding them in contact with the tear film at the medial canthus until the filter From the Bascom Palmer Eye Institute, Depart­ ment of Ophthalmology, University of Miami School of Medicine, Miami, Florida. This study was supported, in part, by Public Health Service Research Grant EY-00031 from the National Eye Institute, and a Research Professorship awarded by Research to Prevent Blindness, Inc. (Dr. Ander­ son). * During this study, Miss Friedland was a student at Coral Gables Senior High School in the Labora­ tory of Research Program of Dade County Public Schools. Presently, she is a Faculty Scholar in the Department of Chemistry, Florida Atlantic Univer­ sity. Reprint requests to Douglas R. Anderson, M.D., Bascom Palmer Eye Institute, 1638 N.W. 10th Av­ enue, Miami, Florida 33152.

paper was saturated. An effort was made to minimize direct contact with the conjunctiva so that tears formed by basal secretion rather than reflex tearing could be obtained. For spectrophotometric analysis and for electrophoresis, tears were collected in a sim­ ilar manner, except that Week cellulose sponges were used to collect a greater quan­ tity of tears. Tear samples from each individual were subjected to a spectrophotometric assay for lysozyme, measurement of total antibacterial activity on agar plates, and measurement of antibacterial activity after mild heating to destroy NLAF. Tear samples from each in­ dividual were also subjected to electrophore­ sis, and individual fractions were subjected to the same tests used on whole tears. To en­ sure consistency of results, determinations were done on each individual on three to five separate occasions. Pooled samples were subjected to dialysis, Sephadex gel filtration, and immunoelectrophoresis. Hen's egg lysozyme ( H E L ) at standard concentrations ranging from 10 to 0.01 mg/ ml were used as controls On each occasion when determinations were made, at least one control concentration of H E L was included to ensure uniformity of the determination from one day to another. Assay for lysozyme—A standard method for assay of lysozyme activity, as described by Sugar, 4 was used. In this method, the bac­ teria, Micrococcus lysodiekticus, whose cell wall is unusually sensitive to the action of ly­ sozyme, is used. Lysis of the bacteria by ly­ sozyme is observed by means of spectrophotometer at a wavelength of 450 ηιμ. Lypholized (freeze-dried) M. lysodiekticus is sus­ pended in 0.1 M phosphate buffer at pH 6.24 in such an amount that the optical density is 0.6 to 0.7 at a wavelength of 450 πιμ. The substance to be assayed is added, and a unit 52

VOL. 74, NO. 1

NON-LYSOZYME ANTIBACTERIAL FACTOR

of lysozyme activity is defined5 as the amount that causes a decrease in optical den­ sity at 450 πιμ of 0.001/minute at pH 6.24

and 25°C. Total antibacterial activity—A lysoplate method modified from the technique of Bonavida and Sapse6 was used. This method is sometimes used as assay for lysozyme based on the assumption that no other antibacterial factor is present. In.our experiment, blood agar plates were inoculated with M. lysodiekticus in a suspension of soy broth with an optical density at 450 πιμ of 0.6 to 0.7 com­ pared to the broth alone. Disks of filter pa­ per 7 mm in diameter were saturated with the test substance, and then allowed to dry at 40°C for 30 minutes. The disks were then place on the freshly inoculated agar plates and incubated at 37°C. After 48 hours, the diameter of the zone of inhibited growth was measured. In the initial stages of the study, a wide range of H E L concentrations was tested to establish a relationship between concentration of lysozyme and diameter of the zone of inhibited growth (Fig. 1). In the later stages of the study when tears and tear

53

fractions were evaluated, a standard concen­ tration of H E L was included in each run of agar plates to ensure reproducibility of the method. Antibacterial activity after heat—Based on the report of Thompson and Gallardo,7 who described the presence of a heat-sensi­ tive non-lysozymal antibacterial factor in tears, the same lysoplate determination was done after heating the tear sample in a man­ ner known to destroy lysozyme. The tears were acidified with one drop of 1N HC1 and heated to 100°C. The pH was normalized with one drop of IN NaOH, and neutrality was confirmed with pH paper. Filter paper disks saturated with the heated tears were analyzed for antibacterial activity in the same manner as described in the preceeding section. Disks saturated with H E L solution of 5 and 10 mg/ml concentrations were used as controls. Electro phoretic separation—The cellulose acetate electrophoresis techniques devised for separation of haptoglobin protein at Jackson Memorial Hospital were modified for use with tears. 8 This technique uses a

20_ E E

!2 «Λ

S < Ξ

.001

.01 HEL

CONCENTRATION, mg/ml

Fig. 1 (Friedland, Anderson, and Forster). Diameter of zone of lysis on lysoplate for various concen­ trations of HEL.

54

AMERICAN JOURNAL OF OPHTHALMOLOGY

small cell and requires only two lambda of sample. The tests were run at a pH of 7.1 for 20 minutes. Initially, samples of H E L and serum were included in each electrophoretic run. The use of H E L pinpointed the position of tear lysozyme on membrane, as it has been shown that H E L migrates electrophoretically to the same position as tear lyso­ zyme.9 Later, serum and H E L controls were replaced with egg white, which contains HEL. Analysis of electrophoretic fractions—To obtain material for analysis from individual electrophoretic bands, membranes were cut in half through the separated fractions and one half was stained for reference. The antibacterial activity in each fraction was determined by cutting out portions of the unstained half according to the reference half. The pieces of membrane containing the various fractions were placed on agar plates inoculated with M. lysodiekticus (lysoplate technique) as described in a previous sec­ tion. The zone of growth inhibition was measured after 48 hours. After it had been determined that one of the electrophoretic bands with a migration different from lysozyme had antibacterial ac­ tivity, material from this band was collected from several samples and eluted by placing the excised portion of the membrane in a tube with 1 ml (0.1 M phosphate) buffer at pH 5.O. The eluate from several samples was pooled to obtain enough material for study of the NLAF. The eluted fraction containing N L A F was assyed for lysozyme by spectrophotometric assay. The fraction was also subjected to heat in a manner that would destroy N L A F but preserve lysozyme. The fraction was then tested for activity against M. lysodeikticus. The eluted fraction was also studied by immunoelectrophoresis and dialysis. Immunoelectrophoresis—Samples of the eluted fraction containing N L A F were con­ centrated 10 times and studied by immunoe­ lectrophoresis against rabbit serum contain­

JULY, 1972

ing antibody against 27 human serum pro­ teins. Whole tears were similarly studied, and serum was used as a control. Dialysis—The eluted fraction was dialyzed to determine whether the antibacterial activity could be attributed to a protein pres­ ent in the N L A F band, a protein and a lower molecular weight co-factor, or a low-molecu­ lar weight factor that migrated along with other constituents of the N L A F band. Fifty lambda of the eluted fraction was dialyzed against 1000 ml of distilled water for four hours, after which the contents of the dialy­ sis bag were assayed for antibacterial activ­ ity on agar plates. To assay the antibacterial activity of the dialysate, 50 lambda of the eluted fraction was dialyzed against 1 ml of distilled water for four hours and samples both inside and outside the dialysis bag were assayed for antibacterial activity. Samples from outside the bag were also heated and tested for activity on agar plates to insure that there were not some contaminant intro­ duced in the elution procedure. Sephadex gel filtration—To estimate the molecular weight of the NLAF, and to bet­ ter separate it from other constituents of tears, Sephadex gel filtration of tears was carried out in a 20 ml column using sizes G25, G-50, and G-100. One hundred lambda samples were used in each test and 30 frac­ tions containing 0.77 ml were collected. The antibacterial activity of each fraction was as­ sayed on agar plates. By comparing the sepa­ rations of lysozyme and the N L A F on each column, the molecular weight could be esti­ mated. On G-50 and G-100 lysozyme was used as a marker for 7700 molecular weight rather than its true molecular weight because of its abnormal migration on Sephadex, as recorded by Whitaker. 10 Antibacterial spectrum—The relative ac­ tivity of lysozyme and non-lysozyme actibacterial factor against M. lysodiekticus was al­ ready determined in the course of the pre­ ceding experiments. Activity of whole tears, NLAF, and lysozyme against Staphylococ-

VOL. 74, NO. 1

NON-LYSOZYME ANTIBACTERIAL FACTOR

eus epidermis, Staphylococcus albus, Escherichia coli, Pseudomonas, Hemophilus influenza, alpha streptococcus, beta strepto­ coccus, Pneumococcus, Proteus mirabilis, Klebsiellae aerobàcter, diptheroids, and Ba­ cillus subtilis were also tested on agar plates. RESULTS

Spectrophotometric assay for lysozyme— The lysozyme activity of tear samples ranged from 24 to 100 units/ml with an average of 58 units/ml. When a wide range of H E L concentrations was tested, it was found that there was at all concentrations a consistent relationship of 20,000 units of ly­ sozyme activity/mg H E L . This means that the lysozyme activity of tears can be ex­ pressed in terms of the concentration of H E L to which it is equivalent, and this will be useful for comparison with results of other experiments. Table 1 shows the lyso­ zyme activity of the tears of eight normal in­ dividuals in this study expressed in terms of equivalent H E L concentrations. The range is from 0.012 to 0.05 mg H E L / m l of tears with an average of 0.029 mg/ml. Total antibacterial activity—The antibac­ terial activity of undiluted whole tears against M. lysodeikticus was compared with a range of concentrations of H E L on agar plates. The activity of tears is expressed in terms of the concentration of the H E L to which it is equivalent. The range of activity was equivalent to 3.0 to 9.5 mg/ml, with an average activity equivalent to 5.0 mg H E L / ml (Table 1). This activity is 200 times greater than would be expected from the ac­ tual amount of lysozyme present in tears as measured by spectrophotometric assay, which is also expressed in terms of equiva­ lent concentrations of H E L . Antibacterial activity after heating— Tears that had been subjected to heat at 100° C at an acid pH showed minimal activity against M. lysodiekticus. The activity ranged from that equivalent to 0.01 to 0.05 mg H E L /ml, with an average activity equivalent to

55

TABLE 1* COMPARISON OF LYSOZYME ACTIVITY MEASURED BY SPECTROPHOTOMETER WITH ANTIBACTERIAL ACTIVITY ON LYSOPLATES BEFORE AND AFTER HEATING IN EIGHT NORMAL INDIVIDUALS

Subject R.K.F. B.R.F. K.P. D.H. H.B.

J.G.

N.C B.M. 5 mg/ml HEL 10 mg/ml HEL

Spectrophotometer 0.050 0.028 0.025 0.032 0.027 0.021 0.012 0.047 5.000 10.000

Lysoplate After Without Heating Heating 9.5 6.0 5.0 6.0 5.0 3.9 3.5 8.0 5.0 10.0

0.050 0.032 0.023 0.035 0.025 0.017 0.010 0.045 5.0 10.0

* All values expressed as equivalent concentrations (mg/ml) of Hen's egg lysozyme (average of three to five determinations).

0.03 mg HEL/ml. (Table 1). These figures correlate very well with the amount of lyso­ zyme activity measured in the spectrophoto­ metric assay. As expected, the concentra­ tions of H E L subjected to heat at an acid pH showed no change in activity on agar plates. Electrophoretic separations—With the 20minute electrophoresis performed, the tear proteins were separated into three fractions (Fig. 2 ) . One fraction moved toward the

Fig. 2 (Friedland, Anderson, and Forster). Elec­ trophoresis membrane showing migration of pro­ teins in egg white and tears.

56

AMERICAN JOURNAL OF OPHTHALMOLOGY TABLE 2

TOTAL ANTIBACTERIAL ACTIVITY* AND LYSOZYME CONTENT* OF FIRST ELECTROPHORETIC FRACTION

Subject

Lysoplate

Spectrophotometer

R.K.F. N.C. K.P. D.H.

0.043 0.013 0.023 0.030 0.025 0.050 0.025 0.030

0.050 0.012 0.025 0.032 0.021 0.047 0.027 0.028

J.G.

B.M. H.B. B.R.F.

* Expressed as HEL concentration to which ac­ tivity is equivalent.

cathode and had the same electrophoretic mobility as HEL. The other two fractions moved toward the anode. This separation is equivalent to the findings of other work­ ers.11·'12 Antibacterial activity of each fraction against M. lysodiekticus was measured (ly­ soplate technique) and expressed in terms equivalent H E L concentrations. The first fraction, which moved in the direction of the cathode, with the same mobility as HEL, had total antibacterial activity (lysoplate) in the range of 0.013 to 0.05 mg HEL/ml of tears with an average activity of 0.03 mg H E L / ml (Table 2 ) . This activity corresponded to the results of spectrophotometric assay for lysozyme (Table 2). On lysoplates, the cen­ ter band exhibited activity equivalent to 3.5 to 8.0 mg HEL/ml of tears with an average of 5.6 mg/ml Table 2. The third band showed no activity at all on the lysoplate. When the center fraction was eluted from several samples and pooled it was found to have no lysozymal activity by spectrophoto­ metric assay (Table 2 ) , despite its high anti­ bacterial activity on Shirmer lysoplates. When the eluted center band was heated to 100°C at an acid pH it lost antibacterial ac­ tion on agar plates. Immunpelectrophoresis — Immunoelectrophoresis (Fig. 3) using polyvalent serum failed to show the presence of any immunoprotein in the eluted fraction containing

JULY, 1972

NLAF. Control samples of whole tears showed the presence of IgA, IgM, and albu­ min, thus confirming that concentrations were high enough in the samples. Although N L A F had a mobility similar to gamma globulin on cellulose acetate electrophoresis, neither the whole tears nor the eluted frac­ tion containing N L A F exhibited gamma globulin on immunoelectrophoresis. Dialysis—On dialysis against 1000 ml, samples taken from inside the bag showed no antibacterial activity. In dialysis against 1 ml, samples from the dialysate outside the bag showed a great deal of activity, with ly­ soplate diameters of 21.5 and 22.5 mm. The samples from inside the dialysis bag had zones of inhibition with diameters of 14.0 and 15.0 mm. This indicates that the N L A F passes easily through the dialysis membrane and therefore has a low molacular weight (under 10,000). Sephadex gel filtration—On the Sephadex G-25 column, both lysozyme and the N L A F

Fig. 3 (Friedland, Anderson, and Forster). Im­ munoelectrophoresis of NLAF, tears, and serum controls against polyvalent antiserum.

VOL. 74, NO. 1

NON-LYSOZYME ANTIBACTERIAL FACTOR TABLE 3

TOTAL ANTIBACTERIAL ACTIVITY* AND LYSOZYME CONTENT* OF SECOND ELECTROPHORETIC

FRACTION CONTAINING N L A F

Subject

Lysoplate

Spectrophotometer

R.K.F. N.C. K.P. D.H. J.G. B.M. H.B. B.R.F.

8.0 3.5 5.0 5.5 3.5 8.0 4.5 5.5

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

* Expressed as HEL concentration to which ac­ tivity is equivalent.

came off the front of the column, indicating that they are above 5000 in molecular weight.13 On both the G-50 and G-100 col­ umns the N L A F came off after the lyso­ zyme. On the G-50 column the N L A F was completely separated from the lysozyme. These data place the molecular weight of the N L A F between 5000 and 7500. Antibacterial spectrum—As seen in Table 4, H E L even in high concentrations showed little activity against any of the conjunctival inhabitant species tested. The two species ly­ sozyme did act against are both Gram-posi­ tive. In contrast, both the tears and the nonlysozyme antibacterial factor eluted from the

57

second electrophoretic band showed activity against a wide range of conjunctival inhibitants, some of them pathogens. DISCUSSION

The results indicate that tears contain at least two antibacterial factors. One of these is lysozyme, and it has the same electropho­ retic mobility as Hen's egg lysozyme. The amount of lysozyme can be assayed by spectrophotometry. The second factor has a different electro­ phoretic mobility, and it is not merely an­ other type of lysozyme since spectrophotometric assays demonstrated that it had no ability to lyse the cell walls of M. lysodiekti­ cus. Nonetheless, this N L A F has a strong antibacterial effect against the growth of M. lysodiekticus, and it also has activity against many conjunctival inhabitants, few of which are affected by lysozyme even in high con­ centrations. Unlike lysozyme, the antibacte­ rial activity of N L A F is destroyed by heating to 100°C at an acid pH, and is thus the same factor previously studied by Thompson and Gallardo.2 The presence of an antibacterial factor that is more powerful than lysozyme and active against a wide spectrum of con­ junctival inhabitants suggests that lysozyme does not serve to kill bacteria in the tear film.

TABLE 4 COMPARISON OF SPECTRUM ACTIVITY OF TEARS, NLAF, AND HEL

E. coli Pseudomonas sp. H. influenza Alpha streptococcus Beta streptococcus Pneumococcus Proteus mirabilis Kl. aerobacter Diptheroids B. subtilis Staph. aureus Staph. epidermis Staph. albus

Tears*

NLAF*

Lysozyme (10 mg/ml)

9.8 19.5 21.0 15.2 13.5 18.3 11.3 21.4 17.3 16.5 9.0 9.5 11.3

9.7 19.8 21.0 15.2 13.6 18.5 11.5 22.5 17.0 16.6 8.5 9.0 11.5

No activity No activity No activity 7.7 mm No activity No activity No activity No activity 7.7 mm No activity No activity No activity No activity

* Figures are diameter (in mm) of zone of growth inhibition.

58

AMERICAN JOURNAL OF OPHTHALMOLOGY

This opens the way to speculation about ly­ sozyme's function in tears. The presence of N L A F also has implica­ tions on the use of the Shirmer lysoplate method as a means of measuring the lyso­ zyme content in tears. Clearly such a tech­ nique measures all antibacterial activity against M. lysodiekticus and is a measure of lysozyme content only if it is known that an­ tibiotics and all other antibacterial factors are absent. In contrast, spectrophotometric assay techniques measure only the lysis of bacteria and are not affected by the presence of other antibacterial factors that operate by some mechanism other than lysis of the bac­ terial cell wall. Electrophoresis remains as another valid means of measuring lysozyme in human tears, since it is now well estab­ lished that the fraction of human tears that has the same electrophoretic mobility as H E L is indeed lysozyme. It should be noted, however, that in other species, tear lysozyme. has a different electrophoretic mobility or of­ ten is absent.14 Sephadex gel filtration results place the molecular weight of N L A F between 5000 and 7500, assuming that N L A F has no ab­ normal affinity for the sephadex column.10 The fact that the N L A F passed through the dialysis bag confirm that it has a molecular weight under 10,000, too small to be the pro­ tein stained material on the electrophoresis membrane. Thus the N L A F is a small mole­ cule that merely has the same electrophoretic mobility as one of tear proteins. Interestingly, the protein that accompanies N L A F on elec­ trophoresis is apparently not one of the com­ mon immunoglobulins even though its elec­ trophoretic mobility is close to that of gamma globulin. Additional studies are needed to define further the nature of NLAF. SUMMARY

A non-lysozyme antibacterial factor was found in normal, non-stimulated tears. Lyso­ zyme content was measured by spectrophoto­ metric assay. Total antibacterial activity was

JULY, 1972

measured in terms of a zone of inhibited growth on an agar plate inoculated with M. lysodiekticus, and it was discovered that total antibacterial activity was 200 times greater than could be attributed to the amount of ly­ sozyme present. Electrophoresis separated the lysozyme from the non-lysozyme antibacterial factor. The latter had no lysozyme activity by spec­ trophotometric assay, but exhibited antibac­ terial activity very close to the total tear ac­ tivity on agar plates. This factor is destroyed by heating to 100°C in an acid medium, whereas lysozyme is not. Dialysis and Se­ phadex gel filtration separated the factor from other constituents of tears and approx­ imated the molecular weight as between 5000 and 7500. The non-lysozyme antibacterial factor exhibits activity against a wide spec­ trum of conjunctival inhabitants including pathogenic species, whereas lysozyme even in high concentrations, does not. ACKNOWLEDGMENTS

Drs. Thomas Noto, Joerg Jensen, P. L. Whitney, and David Anderson generously gave advice and al­ lowed the use of their laboratory equipment for various aspects of this study. We are also indebted to Mr. James Miley of the Laboratory Research Program. REFERENCES

1. Fleming, A. : On a remarkable bacteriolytic el­ ement found in tissues and secretions. Proc. Roy Soc. 93:306, 1922. 2. Thompson, R., and Gallardo, E. : The antibac­ terial action of tears on staphylococci. Am. J. Ophth. 24:635, 1941. 3. Phillips, D. C. : The three-dimensional struc­ ture of an enzyme molecule. Scientific American 215:64, 1966. 4. Shugar, D. : Method of assay for lysozyme (Muramidase). Biochim. Biophys. Acta 8:302, 1952. 5. Mattenheimer, H. : The Theory of Enzyme Tests. New York, Boehringer, Manheim Corp., 1968, p. 8. 6. Bonavida, B., and Sapse, A. T. : Human tear lysozyme. II. Quantative determination with stan­ dard Schirmer strips. Am. J. Ophth. 66:70, 1968. 7. Thompson, R., and Gallardo, E. : The antibac­ terial action of tears on staphylococci. Am. J. Ophth. 24:635, 1941. 8. Noto, T. : Electrophoresis Manual. Miami,

VOL. 74, NO. 1

NON-LYSOZYME ANTIBACTERIAL FACTOR

Florida, Jackson Memorial Hospital, 1970, pp. 1, 52. 9. Bonavida, B., Sapse, A. T., and Sercarz, E. E. : Human tear lysozyme. I. Purification, physiochemical, and immunochemical characterization. J. Lab. Clin. Med. 70(6) :951, 1967. 10. Whitaker, J. R. : Determinations of molecular weight of proteins by gel filtration on Sephadex. Anal. Chem. 35:1950, 1963. 11. Tapaszto, I., and Vass, Z. : Demonstration of

59

the protein factions of human tears by means of micro-electrophoresis. Acta Ophth. 43:796, 1965. 12. Krause, E. : A paper electrophoretic study on human tear proteins. Acta Ophth. 53 (Suppl.) : 1959. 13. Sephadex gelfiltrationin theory and practice. Uppsala, Sweden, Pharmacia, 1966, p. 10. 14. Allansmith, M. R., Drell, D., Anderson, R. P., and Neuman, L. : Tear lysozyme in man. Am. J. Ophth. 71:525, 1971.

O P H T H A L M I C MINIATURE

Temporal indifference: This is the assumption that temporal changes are unimportant in scientific observation and experimentation. By tem­ poral, I refer to biological clocks rather than the fluxion of time inde­ pendent of processes and events. There are a number of biological rhythms ranging in time from a few seconds to seasons. Rhythms are still ignored in many research studies, so that testing experimental groups at one time and the controls at another may merely reveal naturally oc­ curring changes unrelated to the putative causes under study. Another form of the fallacy is failure to properly relate cumulative factors to time. ". . . suppose each punishment was to be going without a dinner. Then, when the miserable day came, I should have to go without 50 dinners at once !" Dwight J. Ingle Fallacies and errors in the Wonderlands of Biology, Medicine, and Lewis Carroll Perspectives in Biology and Medicine 15:271, 1972