PROTEINS IN TEARS FROM HEALTHY AND DISEASED EYES
ARON ZAVARO, ZEMIRA SAMRA, ROBERT BARYISHAK & DAVID SOMPOLINSKY
(Tserifin, Ramat-Gan, lsrael)
Keywords: Tears; Albumin; IgG; IgA; IgM; Crossed immunoelectrophoresis; Normal values; Conjunctivitis vernalis; Conjunctivitis follicularis; Stevens Johnson's syndrome; Blepharoconjunctivitis; Conjunctivitis sica
The levels of total protein in tears from healthy donors, conjunctivitis vernalis patients, and conjunctivitis follicularis patients, were 625, 1370 and 1160 mg% respectively. Serum albumin accounted for 3.3%, 43% and 67% of the total protein of tears from these groups, and the level of proteins probably synthesized by the lacrimal gland, was in tears from conjunctivitis foUicularis patients only half the level in normal tears. By crossed immunoelectrophoresis with intermediate gel, 10 antigenic species could be recognized in normal tears, and of these the following were identified: Lysozyme, IgA, lactoferrin and serum abumin. In tears from patients with conjunti- vifis vernalis three more immunoprecipitates were observed, of which one was due to IgG. No lysozyme could be demonstrated in tears from a case of conjunctivitis sicca by immunoelectrophoresis.
In tears from healthy donors the mean level of IgA was 20 mg%, of IgG 3 rag%, and IgM could not be demonstrated. Rabbit anti-tear immunoglobulin did not precipitate a standard of human IgM in double immunodiffusion. In cases of conjunctivitis ver- nalis and folliculafis the mean levels were increased to 80 and 114 rag% IgG, and 11 and 14 rag% IgM, but IgA was increased only to 32 and 41 rag%. It is assumed that the level of IgA in normal tears is almost entirely due to local synthesis, while serum albumin and other immunoglobulins may have escaped from the circulation by mole- cular sieving. The increased levels of immunoglobulins in inflammatory diseases is probably due to transudation. However, in blepharoconjunetivifis patients several tear samples with a high IgM and a low or zero level of IgG could be demonstrated. Possible explanations for this phenomenon are discussed.
Many investigators have studied the protein components of tears under phy- siological and pathological conditions, but several problems have remained unsettled, partly due to diverging reports. Most authors have studied the le- vels oflysozyme (Ronen et al., 1975; Eylan et al., 1977) or of various immuno- globulins (McClellan et al., 1973; Sen & Sarin, 1979; Little, Centifanto
& Kaufman, 1969; Allansmith, 1973). It is generally assumed that the IgA and IgE of tears are, at least in part, synthesized locally by lymphocytes in the gland tissue, whereas IgG and IgM may have escaped from the blood cir- culation (Allansmith, 1973), but some authors have indicated the possibility of local production of IgG as well, in order to explain the ratio between va- rious immunoglobulins in tears from normal subjects, which often vary from the ratio found in blood plasma (McClellan et al., 1973).
In the present study we have determined the levels of IgA, IgG and IgM in
Documenta Ophthalmologica 50,185-199 (1980). 0012-4486/80/0501-0185 $ 3.25. 9 Dr. W. Junk B.V. Publishers, The Hague. Printed in The Netherlands.
tears of healthy and externally diseased eyes. We have paid particular atten- tion to the ratio between these immunoglobulins and serum albumin (HSA). The concentration of HSA in tears is used as a measure for transudation from the circulation from where it probably originates.
MATERIAL AND METHODS
Tears were obtained from 50 healthy donors without a history of ocular diseases. The age of the donors varied from 2 to 70 years; 24 were females and 26 were males. Tear samples were also obtained from 60 patients with well-defined disorders of the external eyes. The tears were drawn into mi- crotubes by capillarity from the right and the left eye separately, always in this sequence, and no stimulation was employed. In a few cases only one sample could be obtained.
Quantitation of proteins
Total protein of tears was determined by a modification of Lowry's method
(Hartree, 1972). HSA and immunoglobulins were quantitated by radial im- munodiffusion in 1% agarose (Mancini, Barbonara & Heremans, 1965). Rab- bit antisera against serum albumin and human heavy chain immunoglobu- fins (a, 7 and/~, respectively) were used. Four #1 of the tears or of protein standards were introduced into wells in the antiserum containing agarose. Incubation was 24 hours at 37~ (for HSA, IgG and IgA) and 96 hours (for IgM). For each protein two gels were used, one with 2/A antiserum per cm 2 , the other with 0.2 ~tl per cm 2. In this way linear calibration curves in the intervals ~>0.3 - 100 rag% (HSA, IgG and IgA) or/>0.6 - 100 rag% (IgM) were obtained. After washing, the gels were inserted into 1% tannic acid for sharp visualization of the zones of immunoprecipitation. Serum and secre- tory IgA were not differentiated.
Immunization of rabbits
Rabbits were immunized by weekly subcutaneous injections of a pool of equal parts of 24 tear samples from healthy donors. 0.5 ml of the tears was mixed with 0.5 ml incomplete Freund's adjuvant. Rabbit immunoglobulin was obtained from serum by sedimentation with 50% (NH4)2 SO4 and dia- lysis of the redissolved sediment against 10 mM tris (hydroxymethyl) ami- nomethane, 0.5 M NaC1,0.1% NAN3, pH 7.2.
Crossed immunoelectrophoresis with intermediate gel and tandem crossed immunoelectrophoresis in agarose were performed as described by Axelsen (1973) and Bock & Axelsen (1973). The second dimension agarose gel con- tained 20 #l/cm 2 rabbit anti-human tear immunoglobulin. After electro- phoresis, the plates were washed in 50 mM barbital buffer at pH 8.6 and thereafter in distilled H20. Staining was with Coomassie Blue R250. For some of the immunoprecipitates obtained by crossed agarose electropho- resis the corresponding antigen could be identified, as will be specified un- der Results. The following techniques were used to identify the antigens, except for lysozyme: 1. Tandem-crossed immunoelectrophoresis (Bock & Axelsen, 1973), with tears in one well and a known protein standard in the second well. By immunological identity the immunoprecipitate of the known protein merges with the corresponding precipitate of the tear sample. 2. A known protein is added to the tear sample before crossed agarose electrophoresis. The immunoprecipitate corresponding to this protein is enlarged. 3. A monospecific antiserum to a known protein is incorporated into the intermediate gel of the crossed electrophoresis (Axelsen, 1973). The corresponding immunoprecipitate is drawn into the intermediate gel.
Demonstration of lysozyme
The electrophoretic mobil ity of lysozyme was determined by electrophore- sis on a agarose strip similar to the first dimension of crossed agarose electro- phoresis. The gel was thereafter dried at 40~ and overlayed with a 1.5 mm- thick agarose layer, which contained 3 mg of dried Mierocoeeus lyseidecti- cus. After incubation for two hours at 37~ a sharp zone of bacteriolysis indicated the lysozyme.
Ouchterlony double immunodiffusion
The antigens and rabbit immunoglobulins were applied in 8/~1 amounts in- to wells in the 1% agarose gel and the plates were incubated for 48 hours at 37~ thereafter washed and stained as for crossed agarose electrophoresis.
HSA and rabbit anti-HSA immunoglobulin were from Dakopath AS, Den- mark. Human immunoglobulin standards and rabbit anti-heavy chain immu- noglobulins, transferrin, antitransferrin and antilactoferrin were purchased from Behring AG, Germany.
Analyses for mean, standard deviation and significance of differences (p) between the groups by the unpaired t-test were performed on a computer- ized program.
The source o f tear proteins
Table 1 shows that serum albumin (HSA) accounted for 3.3%, 43% and 67% of total protein in tears from healthy donors, and patients with conjunctivi- tis vernalis and follicularis, respectively. In cases of conjunctivitis sicca we did not obtain enough fluid for determination of total protein. We have therefore washed the conjunctival sac thoroughly and aspirated a part of the wash fluid. In this case HSA accounted for 77% of total protein. The le- vel of HSA was 20, 590 and 773 rag% in the pools of normal tears, tears from conjunctivitis vernalis and from conjunctivitis follicularis, respectively. These figures reflect probably an increasing degree of transudation of circu- latory proteins. The Table shows also that the level of proteins other than HSA and immunoglobulins were similar in tears from healthy persons and patients with conjunctivitis verualis, in both groups about 600 rag%, but it was only 271 rag% in conjunctivitis follicularis.
Table 1. Total protein, serum albumi~ and immunoglobulins in tears.
Total Serum IgA lgG IgM protein 1 albumin
Pool of tears from 24 healthy 625 20.5 23.5 0.93 ~--0.6 donors (3.3%) 2 (3.7%) (0.15%)
Pool of tears from 10 patients 1370 590 26 68 22 with Conjunctivitis vernalis (43%) (2%) (5%) 1.6%)
Pool of tears from 9 patients with 1160 773 42 64 9.9 Conjunctivitis folticularis (67%) (3.6%) (5.5%) (0.8%)
Sample from Conjunctivitis sicca 3 380 292 11 47 5 (77%) (3%) (12%) (1.4%)
1 All values are in mg%. 2The figures in brackets indicate percentage of total protein. 3No tears could be obtained. The right conjunctival sac was thoroughly washed with sterile saline and 40/31 of the fluid was aspirated into a mierocapillar. The figures are relative only.
Proteins in tears from healthy eyes
A pool of equal parts of 24 samples of tears from healthy donors was exa-
mined by crossed agarose electrophoresis with anti-tear immunoglobulin. Ten immunoprecipitates could be clearly distinguished, though one of them
(3a) did not always stay clearly out on the photo (see Figs. 1 and 2). Of the- se, the two most voluminous ones correspond to lysozyme (No. 9) and a protein with fast anodal mobility (No. 1), probably the low-molecular weight acidic protein described by Josephson and Weiner (1968) and Bonavida, Sapse and Sercarz (1969). Precipitate No. 8 was shown to be due to IgA and No. 3 to be serum albumin (Fig. 2). Precipitate No. 7 was likewise shown to be due to lactoferrin. In double immunodiffusion the anti-tear immunoglo- bulin reacted strongly with standards of human IgG, but not with IgM or transferrin.
In Table 2 are compiled determinations of HSA and immunoglobulins of tear samples from healthy and diseases eyes, and Table 3 shows the signifi- cance of differences between the groups (p) as obtained by a computerized program of the unpaired t-test. These tables are based on samples from the right eye only. The results of tears from the left eyes have been examined statistically in the same way and the mean values and degrees of significan- ce were almost identical.
Fig. 1 (Zavaro, Samra, Baryishak, Sompolinsky). Crossed agarose immunoelectropho- resis with intermediate gel of tear fluid from healthy eyes. The sample was 5 #1 of a pool of equal amounts of tear fhLid from 24 healthy persons. The application slit is indicated by an arrow. 20 #l]cm 2 rabbit anti-tear immunoglobulin was added to the second dimension gel. Immunoprecipitate No. 3 has been identified as due to serum albums, No. 7 as lactoferrin, No. 8 as IgA and No. 9 as lysozyme. Precipitate 3A (see Fig. 2) is not clearly seen on the photo. The anodal electrode to the right and the top in first and second dimension electrophoresis, respectively.
Fig. 2 (Zavaro, Samra, Baryishak, Sompolinsky). Identification of serum albumin crossed immunoelectrophoresis with intermediate gel of tears, a. 5//1 of normal tears. b. 5 /21 tears + 5 pl serum albumin, 90 pg/ml. Immunoprecipitate No. 3 is strikingly in- creased, c and d. Tandem-crossed electrophoresis. In c, 5/11 tears were applied into the left and in d, into the right well. Into the other wells was applied 5/11 of 90 pg/ml se- rum albumin. The immunoprecipitate of serum albumin merged with precipitate No. 3. The anode to the right and the top of the figures. T = tears A = serum albumin
In tears from healthy donors HSA levels varied in the range 5-27 mg%, IgA levels in the range 13-25 rng% and the highest level of IgG observed was 12 mg%, but about 50% of the samples were negative for IgG (
+I +1 +1 +1 +1
v = 4 d d d
oh O0 o'~
+1 +1 +1 +1 +1 +1
~ O0 O~
+1 +1 +1 +1 +1 +1 ~- ~ o~ o. ~,
+1 +1 +1 +1 +1 +1
9 ~ .~ .~:~ ~ ~ , .~- o o o 0~'
,~-= ~ "~ '= .~, o~ ~ ~ ~.~ 0 o 0 0 0
that HSA and IgG in the tears come from the circulation through a blood- tear barrier which retains high molecular weight proteins most effectively. The results were analyzed statistically for the correlation with age, sex and right versus left eye, but no significant correlation was found.
Tears from externally diseased eyes
Fig. 3 shows a crossed immunoelectropherogram of a pool of 10 tear samples from conjunctivitis vernalis against immunoglobulin to normal tears. All the immunoprecipitates from Fig. 1 were recognized, and only No. 3 (HSA) was strongly altered in size. The identity with the corresponding antigens in nor- mal tears was demonstrated by tandem-crossed immunoelectrophoresis. Three precipitates, not observed in Fig. 1, occurred on the electrophero- gram of tears from conjunctivitis vernalis patients, one of which (C in Fig. 3) was shown to be due to IgG. The lowest concentration of IgG observed in this group was 19 mg%, which is higher than the highest level in the group of healthy eyes. The mean value for HSA (600 rag%, see Table 2) was 37 times
Fig. 3. (Zavaro, Samra, Baryishak, Sompolinsky). Crossed immunoelectrophoresis with intermediate gel of 5/dl of a pool of tears from patients with conjunctivitis verna- lis. Immunoprecipitate NO. 3 is strikingly increased. Three immunoprecipitates not observed in Fig. 1 are designated A, B and C. C was shown to be due to IgG. The anode at the right and the top.
- and the mean IgG (81 mg%) was 26 times as high as in the healthy group, but the mean IgA was only 1.6 times as high. IgM varied between 0.5; still, the t-test showed that the distribution of concentrations of HSA, IgA, IgG and IgM, was signifi- cantly different from the group of healthy donors (p
From five patients with keratoconjunctivitis sicca we obtained tears (Ta- bles 2 and 3). The highest HSA level was 3100 rag%, which is the lower limit of normal plasma concentration. The values for HSA, IgA, IgG and IgM in tears of conjunctivitis sicca patients were significantly higher than in tears from healthy donors and from patients with conjunctivitis vernalis (except for IgG), but not significantly different from the samples of conjunctivitis follicularis (Table 3). The Alb/G ratio was 14.6 and the G/M ratio 3.7. Crossed im...