Complement proteins and C3 anaphylatoxin in the tears of patients with conjunctivitis Mark Ballow, M.D., Peter C. Donshik, M.D., and Louis Mendelson, M.D. Farmington, Conn. Previous studies in our laboratory have demonstrated pollen-specific IgG antibodies in the tears of patients with vernal conjunctivitis (VC) and elevated tear IgG levels in patients with contact lens-induced giant papillary conjunctivitis (GPC). Tear secretions were examinedfor complement (C) proteins to determine the role of this effector system in the pathogenesis of these ocular disorders. The tears of VC (15) and GPC (10) patients with active disease had elevated tear levels of both C3 and factor B. By use of transferrin as a marker for the leakage of plasma proteins into the tears, most C3 was locally produced by the conjunctival tissues. Although immune complexes could not be detected in the tear secretions, increased levels of C3 des Arg were present in the tears that suggested complement activation with the generation of anaphylatoxins. These studies suggest that complement may be important in the injlammatory ocular process of VC and GPC and that the generation of anaphylatoxins (C3a), even by nonimmune mechanisms, may contribute to basophil and mast cell activation with the release of injlammatory mediators into the tear secretions. (J ALLERGY CLIN IMMUNOL76:473-6, 1985.) The clinical features and histopathologic findings in VC and GPC suggest that the pathogenesis of both ocular diseases has a complex immunologic basis.’ In support of this hypothesis, our laboratory has dem- onstrated pollen-specific IgE and IgG antibodies in the tears of VC patients’. ’ and more recently, elevated levels of IgE and IgG in the tears of patients with GPC.4 Since both ocular diseases are considered by some investigators, at least clinically, to be an IgE-mediated disease,‘.’ the role of the elevated tear IgG and the IgG antibodies in the pathogenesis of these eye dis- eases is unclear. Several groups have detected C pro- teins, e.g., C3, C4, and factor B, in the tears of normal subjects.‘-” IgG may act in concert with the C system to protect the ocular surface or in contrast, interact to generate biologic products that contribute to the in- flammatory process.” This study was undertaken to determine the levels of several C components and the presence of C products of potential biologic impor- From the Departments of Pediatrics and Surgery, Division of Oph- thalmology, University of Connecticut Health Center, Farming- ton, Corm. Supported by Grants EY-2036 and EY-5189 from the National Eye Institute of the United States Public Health Service, the Uni- versity of Connecticut Research Foundation, and the Solomon Eye Fund. Received for publication Sept. 28, 1984. Accepted for publication Jan. 9, 1985. Reprint requests: M. Ballow, M.D., Department of Pediatrics, Uni- versity of Connecticut Health Center, Farmington, CT 06032. Abbreviations used VC: Vernal conjunctivitis GPC: Contact lens-induced giant papillary con- junctivitis C: Complement C3a: C3 anaphylatoxin C5a: C5 anaphylatoxin tance in tears. Increased levels of C3, factor B, and C3a anaphylatoxin were present in the tears of patients with VC and GPC with active disease. These studies suggest that C may play a role in the inflammatory ocular process of VC and GPC. METHODS AND SUBJECTS Fifteen patients with VC and 10 patients with active GPC were studied. The diagnosis of VC and GPC was based on typical symptoms and physical findings as outlined previ- ou~ly.‘~~ Five patients with asymptomatic GPC, but who still had conjunctival papillae on physical examination, were also studied. These GPC patients received new contact lenses and had tears collected at the 3- to 6-month follow- up visit. Control groups included 11 patients with allergic conjunctivitis, seven normal individuals, and 11 normal sub- jects who wear soft contact lenses. Methods Serum was obtained by venipuncture and stored at - 70” C. Tears were collected into glass capillary tubes as pre- viously described.2 One hundred microliters to 300 ~1 were 473
Transcript
Complement proteins and C3 anaphylatoxin in the tears of patients with conjunctivitis
Mark Ballow, M.D., Peter C. Donshik, M.D., and Louis Mendelson, M.D. Farmington, Conn.
Previous studies in our laboratory have demonstrated pollen-specific IgG antibodies in the tears of patients with vernal conjunctivitis (VC) and elevated tear IgG levels in patients with contact lens-induced giant papillary conjunctivitis (GPC). Tear secretions were examinedfor complement (C) proteins to determine the role of this effector system in the pathogenesis of these ocular disorders. The tears of VC (15) and GPC (10) patients with active disease had elevated tear levels of both C3 and factor B. By use of transferrin as a marker for the leakage of plasma proteins into the tears, most C3 was locally produced by the conjunctival tissues. Although immune complexes could not be detected in the tear secretions, increased levels of C3 des Arg were present in the tears that suggested complement activation with the generation of anaphylatoxins. These studies suggest that complement may be important in the injlammatory ocular process of VC and GPC and that the generation of anaphylatoxins (C3a), even by nonimmune mechanisms, may contribute to basophil and mast cell activation with the release of injlammatory mediators into the tear secretions. (J ALLERGY CLIN IMMUNOL 76:473-6, 1985.)
The clinical features and histopathologic findings in VC and GPC suggest that the pathogenesis of both ocular diseases has a complex immunologic basis.’ In support of this hypothesis, our laboratory has dem- onstrated pollen-specific IgE and IgG antibodies in the tears of VC patients’. ’ and more recently, elevated levels of IgE and IgG in the tears of patients with GPC.4
Since both ocular diseases are considered by some investigators, at least clinically, to be an IgE-mediated disease,‘.’ the role of the elevated tear IgG and the IgG antibodies in the pathogenesis of these eye dis- eases is unclear. Several groups have detected C pro- teins, e.g., C3, C4, and factor B, in the tears of normal subjects.‘-” IgG may act in concert with the C system to protect the ocular surface or in contrast, interact to generate biologic products that contribute to the in- flammatory process.” This study was undertaken to determine the levels of several C components and the presence of C products of potential biologic impor-
From the Departments of Pediatrics and Surgery, Division of Oph- thalmology, University of Connecticut Health Center, Farming- ton, Corm.
Supported by Grants EY-2036 and EY-5189 from the National Eye Institute of the United States Public Health Service, the Uni- versity of Connecticut Research Foundation, and the Solomon Eye Fund.
Received for publication Sept. 28, 1984. Accepted for publication Jan. 9, 1985. Reprint requests: M. Ballow, M.D., Department of Pediatrics, Uni-
versity of Connecticut Health Center, Farmington, CT 06032.
tance in tears. Increased levels of C3, factor B, and C3a anaphylatoxin were present in the tears of patients with VC and GPC with active disease. These studies suggest that C may play a role in the inflammatory ocular process of VC and GPC.
METHODS AND SUBJECTS
Fifteen patients with VC and 10 patients with active GPC were studied. The diagnosis of VC and GPC was based on typical symptoms and physical findings as outlined previ- ou~ly.‘~~ Five patients with asymptomatic GPC, but who still had conjunctival papillae on physical examination, were also studied. These GPC patients received new contact lenses and had tears collected at the 3- to 6-month follow- up visit. Control groups included 11 patients with allergic conjunctivitis, seven normal individuals, and 11 normal sub- jects who wear soft contact lenses.
Methods
Serum was obtained by venipuncture and stored at - 70” C. Tears were collected into glass capillary tubes as pre- viously described.2 One hundred microliters to 300 ~1 were
473
474 Ballow et al. J ALLERGY CLIN. IMMUNOL. SEPTEMBER 1985
TABLE I. Tear levels of C3 and factor B in patients with VC and GPC
c3 Factor B (pglmll (pg/ml)
Normal subjects (7) 4.3* t 1.1 co.041 Control contact lens wearers (11) 4.5 k 1.4 0.63 k 0.07 Allergic conjunctivitis (11) 3.1 IfI 1.4 0.77 2 0.26 vc (15) 29.8 +- 7.9 p < 0.001t 6.9 k 2.2 p < 0.01t GPC. active (IO) 17.3 -t 7.6 p < 0.05 2.2 ? 0.6 p < 0.005$ GPC. inactive (5) 4.5 k 1.2 NS 2.2 +- 0.8 p < O.Ol$
NS = not significant. “Mean -t SEM. thdent’s t test comparison to normal subjects. $&dent’s t test comparison to control contact lens wearers.
TABLE II. Tear levels of C3a des ARG
C3a Patient group by/ml) p value
Allergic conjunctivitis (11) 6.9* 2 4.4 vc (10) 381 k 101 <0.0011 GPC, active (I I) Ill ? 25.3 <O.OOl t GPC, inactive (5) 11 k 8.5 NSt
NS = not significant. *Mean t SEM. :Student’s t test comparison to allergic conjunctivitis group.
collected and placed into conical polypropylene microcen- trifuge tubes containing disodium EDTA. The final concen- tration of the EDTA was estimated at 0.01 to 0.02 mol/L. The tears were stored frozen at -70” C. Tear IgE levels were measured by the RAST method (Pharmacia Diagnos- tics, Piscataway, N. J.) IgG, C3, and factor B were mea- sured in the tears by ELBA3 by use of monospecific antisera reagents (N. L. Cappel Laboratories, Inc., Cochranville, Pa.). Transferrin was used as a marker for the leakage of plasma proteins into the tear secretions’, ’ by use of the methods and formulas of Mathews” and Deuschl and Jo- hannson.” Transferrin was measured in tears and serum by radial immunodiffusion.
C3a des Arg and C5a des Arg were quantitated in the tears by radioimmunoassay by use of the methods of Chen- oweth and Hugh” with reagents from Upjohn Diagnostics (Kalamazoo, Mich.). Tears were assayed for Clq binding immune complexes by a solid phase Clq binding ELISA method” and for C3 containing immune complexes by an ELISA method as described by Pereira et al.16 Tear secre- tions were diluted 1: 5 for assay. The sensitivity of these methods for the detection of immune complexes is approx- imately 10 pg/ml of aggregated gammaglobulin.
RESULTS
Table I illustrates the tear levels of C3 and factor B. Normal subjects, normal individuals who wear contact lenses, and patients with allergic conjunctivitis
had low levels of tear C3 (4.3, 4.5, and 3.1 p,g/ml, respectively) and unmeasurable or low levels (0.63 and 0.77 p&ml, respectively) of tear factor B. In contrast, patients with VC and GPC had elevated tear levels of both C3 (29.8 kg/ml and 17.3 kg/ml, re- spectively) and factor B (6.9 Fg/ml and 2.2 pg/ml). Patients with inactive GPC had normal tear levels of C3, but the levels of factor B were still elevated.
In order to examine whether C was being activated in the tear secretions with the generation of biologi- cally active products, C3a des Arg and C5a des Arg were measured. Although pysiologically inactive, these C activation products from C3 and C5 are rel- atively stable and, thus, can be measured by radio- immunoassay. I4 As illustrated in Table II, the tears of patients with VC and GPC had markedly elevated levels of C3a des Arg in contrast to patients with allergic conjunctivitis or inactive GPC patients. By contrast, measurable amounts of C5a des Arg (>5 ng/ ml) could be detected in only one of three tear samples in the VC group (7.2 rig/ml) and one of 10 tear sam- ples in the GPC group (18 rig/ml).
Since the generation of these C activation products may have originated from activation of the C system cascade, the tears were tested for immune complexes. Tear samples (N = 10) were tested for both C lq bind- ing and C3 containing immune complexes. Neither assay detected the presence of immune complexes in the tear secretions.
We examined whether the C3 in tears was produced locally by the conjunctival tissues by use of transferrin as a marker for the leakage of plasma proteins into the tears. As illustrated in Table III, most tear C3 in patients with VC (77%) and GPC (90%) was locally produced by the tissues of the external eye.
DISCUSSION
Previous studies in our laboratory*-“ and OtherS’-7. 17, 18 have suggested that IgE-mediated and
VOLUME 76 NUMBER 3
TABLE III. Local ocular production of C3
Complement proteins and C3 anaphylatoxin in tears 475
Double ratio* % Local formula production
Index of teart secretion
vc (IS) 32.Q 71 0.53 6.1 to II50 31 to 98 0.02 to 2.95
GPC (IO) 133 90.5 0.92 32.6 to 303 72 to 98 0.13 to 2.5
*Ratio > I indicates local production.” <iRatio 10. I indicates local production.” *Mean. BRange of’ values.
IgG-mediated immune mechanisms play a role in the pathogenesis of VC and GPC. A recent study by Bal- low et al.’ demonstrated that 76% of VC patients had specific IgG antibodies to pollen antigens in their tear secretions that were locally produced by the conjunctival tissues. The relationship between these specific IgG antibodies and an IgE-mediated immune process in the pathophysiology of VC is not known. Classically, in allergic disorders, IgG may serve as blocking antibodies for IgE-mediated immune reactions. ” Alternative mechanisms include immune complex immunologic reactions with activation of the C system.
The C system is a complex network of proteins, enzymes, and inhibitors that, together, exhibit im- portant biologic activities in host defense and inflam- matory reactions.“” Although C is usually believed to be a plasma protein system, small amounts of C have been detected in a variety of body fluids, including cerebrospinal fluid, synovial fluid, middle ear effu- sions, and ocular fluids and tissues.2’-‘5 With regard to the eye, C has been found in tears and aqueous humor.‘-‘, 25 Chandler et al.’ demonstrated that hemo- lytic C4 was present in the tears of normal subjects. Bluestone et a1.9 detected C3 in the tears of five normal individuals. Recently, Yamamoto and Allansmith”’ have reported the presence of functional hemolytic C, factor B, C4, C3, and C proteins in the tears of normal subjects. Thus, C is present in the tears of normal individuals that may play a role in protecting the ex- ternal eye against infection or participate in inflam- matory reactions that could lead to ocular disease.
Increased tear levels of C3 and factor B were dem- onstrated in patients with VC and active GPC. Fur- thermore, most tear C3 was locally produced by the tissues of the external eye. The latter is not surprising since the histopathology of both VC and GPC dem- onstrates an intense mononuclear cell infiltrate. Stud- ies by WhaleyZ6 and Einstein et al.” have demon- strated that mononuclear phagocytic cells can syn-
thesize a number of C components of both the classical and alternative pathways. The activation of C plays an important role in host defense and the pathophys- iology of certain types of tissue injury. C may be activated by either the classical pathway via IgG or IgM containing immune complexes or through acti- vation of the alternative pathway.” The alternative pathway can be activated by aggregated IgA or IgG or more commonly by certain lipopolysaccharide gram-negative organisms and cell membranes of transformed cells.2y The activation of either C pathway leads to the generation of two important anaphylatox- ins, C3a and C5a. ” C3a causes a noncytolytic release of histamine from mast cells and basophils, contrac- tion of smooth muscle, and an increase in capillary permeability.30, 3’ It is extremely difficult to measure the biologic activity of these anaphylatoxins in body fluids because they are inactivated rapidly by an en- zyme inactivator (carboxypeptidase).32 However, the physiologically inactive products, C3a des Arg and C5a des Arg, can be measured by radioimmunoas- say.14 We could find only C3a des Arg in appreciable amounts in the tears of patients with VC and GPC. The failure to find C5a des Arg probably relates to its very low concentration in tear fluids.
In order to delineate the activation mechanisms re- sponsible for the generation of C3a des Arg, tears were tested for the presence of immune complexes. Although we could not detect the presence of immune complexes in tears by two different methods, these negative findings do not exclude the possibility that tears contain immune complexes in very low concen- trations. For example, the detection of immune com- plexes in tears could be below the level of sensitivity of these two assays or the complexes may have been dissociated by enzymes normally present in tear se- cretions. Nevertheless, our studies suggest that C plays a role in the inflammatory ocular process of VC and GPC. The generation of C3 anaphylatoxins, even by nonimmune mechanisms, may be important in ba-
476 Ballow et al. J. ALLERGY CLIN. IMMUNOL. SEPTEMBER 1985
sophil/mast cell activation with the release of vaso- active mediators into the tear secretions.
We thank Drs. T. Kennedy and A. Theofilopoulos (Scripps Clinic and Research Foundation) for assaying the tears for immune complexes. We also thank M. Marrow, P. Rapacz, and A. Pantschenko for their technical assistance and J. Vivian0 for her secretarial help.
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