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Observations on the Site and Mechanisms of Antigen-Antibody Interaction in Anaphylactic Hypersensitivity*
OBSERVATIONS ON T H E SITE AND MECHANISMS O F ANTIGENANTIBODY INTERACTION IN ANAPHYLACTIC HYPERSENSITIVITY* F.
G. GERMUTH, JR., M.D., L. B. SENTERFIT, M.D.,
A.
E. MAUMENEE, M.D.,
C. E. V A N ARNAM, M.D.,
J.
PRATT-JOHNSON, 1
AND A. D. POLLACK,
M.D., M.D.
Baltimore, Maryland
Previous work from this laboratory has been concerned primarily with the dynamics of antigen-antibody interaction and the rela tionship of this process to the tissue lesions in hypersensitivity of the serum sickness type. These investigations integrated with the work of others may be briefly sum marized. Studies by Hawn and Janeway 1 and our selves2' 3 have demonstrated that the lesions of serum sickness appear as an animal be gins to form antibody while antigen is still present in the circulation and tissue fluids. With continued production of antibody, an tigen is eliminated, free antibody appears in the circulation, and the organic manifesta tions of serum sickness resolve. Since anti gen is present in the blood during the early antibody response, it was suggested by us some years ago 2 - 3 that the tissue lesions might arise secondary to the transport of an tibody by antigen in the form of soluble an tigen-antibody complexes. In the intervening years, Sternberger, et al.,4 and Weigle5 have demonstrated antigen-antibody complexes in the circulation. Mellors, et al.,6 and Dixon, et al.,7 employing fluorescent antigen and anti bodies found antigen and gamma globulin lo calized in the tissue lesions of hypersensitiv ity. It is not known whether the localization of gamma globulin in the tissue represents specifically fixed antibody. Other work from our laboratory has indicated that soluble anti gen-antibody complexes produced in vitro may elicit anaphylactic shock in guinea pigs, produce Arthus reactions in the skin, and in* From the Department of Pathology and the Department of Ophthalmology, The Johns Hop kins University Medical School. t Fight-for-Sight fellow of the National Council to Combat Blindness, New York, 19S7-S8. 282
duce glomerular and splenic lesions of the serum sickness type following infusion into normal rabbits. 8 ' 9 Presumably these reactions require complement. 10 '" There is then consid erable evidence that soluble antigen-antibody complexes exhibit significant biologic activ ity and that they may play a leading role in the development of allergic reactions. It is clear that important strides have been made in an area which may be called the immunogenesis of immediate-type allergic re actions. Two of the most important problems which still demand clarification concern firstly, the mechanism by which antigen-an tibody-complement complexes injure tissue, and, secondly, the site of tissue injury. In regard to the precise biochemical mech anism through which antigen-antibody-com plement interaction results in tissue injury little is known. Studies currently being car ried on by others 12 ' 13 on the role of comple ment in the hemolysis of red blood cells could provide data which might contribute to an un derstanding of tissue damage in allergic reactions. Many workers believe that comple ment is concerned in the activation of an enzymatic system in the presence of reactive antigen and antibody. 12 ' 13 In regard to the site of tissue injury in al lergic reactions, numerous studies have been made. There seems little doubt that anaphy lactic reactions are manifested in the vascu lar and connective tissue systems. Whether damage to the vascular system is primarily an expression of alteration in the connective tissue which is indissolubly associated with blood vessels or whether these changes are due to endothelial injury has never been re solved. Since marked alteration of the con nective tissues is prominent in hypersensitiv ity, the first of these possibilities seems more
ANAPHYLACTIC HYPERSENSITIVITY
acceptable and warrants further investiga tion. Some years ago, Rich and Follis" failed to produce a typical hemorrhagic Arthus re action in the rabbit cornea unless vascularization had been previously induced. They noted "slight edema, slight polymorphonuclear infiltration, and moderate swelling of the corneal fibers" 24 and 48 hours after in jection of horse serum into the avascular cornea of sensitized animals. It would seem that these workers produced a true Arthus phenomenon in the vascularized corneas of sensitized animals. However, this might be expected under the conditions of the experi ment since vascularization of the cornea in effect converts the cornea into a structure very similar to skin. Since the cornea consists of almost pure connective tissue comprised of fibroblasts, collagen, and reticulum fibers, and amor phous "ground substance" far removed from a blood supply, and since there is so much experimental and pathologic evidence of con nective tissue damage in hypersensitivity, it was deemed useful to employ this tissue in further studies of hypersensitivity, using quantitative immunologic methods to see whether connective tissue damage could in deed be related to antigen-antibody interac tion in the absence of blood vessels. Recent work in our laboratory has disclosed that connective tissue change can be produced in the avascular cornea at the site of local anti gen-antibody interaction. These experiments were of two designs. In one type, appropri ate quantities of bovine serum albumin or bovine serum gamma globulin were injected into the avascular cornea of passively or ac tively sensitized rabbits. In the second type of experiment, bovine serum albumin and its corresponding antibody were simultaneously injected into different sites of the avascular corneas of normal rabbits. In all experi ments, alterations in the connective tissue were observed which have been interpreted as those of degeneration or necrosis. Al though these changes were accompanied by
283
acute exudative inflammation, they did not appear to be dependent on leucocyte migra tion since similar changes were noted in ani mals which had been rendered leucopenic by nitrogen mustard injections. In each case, the connective tissue alteration occurred at the site of maximal precipitation of antigenantibody complexes as demonstrated by fluor-marking of the injected antigen. Following the injection of 0.1 ml. of fluor-labeled foreign protein into the center of the cornea of actively or passively sensi tized animals, the limbus appeared grossly inflammed six to eight hours later. From 12 to 24 hours later, a grayish arc of opacification appeared in the periphery of the cornea. It was separated from the limbus by a clear area one to three mm. in width (fig. 1). Further observation for an additional 24 hours generally disclosed completion of the arc to form a concentric ring of opacification. Examination of such eyes under Wood's light in the living animal revealed
Fig. 1 (Germuth, et al.). Living eye in rabbit previously sensitized to bovine albumin. Twentyfour hours after injection of 1.0 mg. fluor-bovine albumin into center of cornea. Note opaque ring in periphery of cornea with clear cornea between ring and limbus. Dilatation and engorgement of limbal and bulbar conjunctiva! vessels with petechial hemorrhages. Under Wood's light the gray opaque ring presented bright green fluorescence. (The two bright spots are highlights.)
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F. G. GERMUTH, JR. et al
Fig. 2 (Germuth, et al.). Transverse section of peripheral portion of cornea and adjacent limbus. Rabbit previously sensitized with bovine albumin. Received injection of 1.0 mg. fluor-bovine serum albumin in center of cornea 30 hours before death. Hemorrhage and neutrophilic leukocytic infiltra tion around limbal vascular loops. Sharp transverse line of leukocytic infiltration and collagen degenera tion distant from limbus. This line corresponds to a section through the opaque ring seen grossly. Note leukocytes migrating from limbus across clear corneal zone to line. No blood vessels are present in cornea.
sharp localization of antigen in the ring of opacification. Histologic examination of corneas show ing such rings (fig. 2) revealed a sharp line of concentrated leukocytes traversing the cornea from Bowman's to Descement's mem brane. The leukocytes had evidently mi grated from the limbal side as evidenced
Fig. 3 (Germuth et al.). Moderate magnification of cornea to illustrate the histologic appearance of the opaque ring. Leukocytic infiltration and swell ing and degeneration of corneal stroma.
Fig. 4 (Germuth et al.). Fluorescence prepara tion of lesion similar to that of Figure 3. The in tense white line represents a bright green line of fluorescence under ultraviolet illumination due to fixation of fluor-antigen by antibody diffusing from limbus in a sensitized animal 30 hours after intracorneal injection 1.0 mg. fluor-bovine serum albu min. The line of fluorescence coincides precisely with the line of leucocyte infiltration and collagen degeneration. (The bright appearance of the cells is due to autofluorescence.)
by the presence of scattered leukocytes be tween the limbus and the sharp line of leu kocytic infiltration. The limbal vessels were dilated and engorged and the loose connec tive tissue around them was infiltrated by leukocytes, red blood cells, and fibrin. Closer examination of the corneal line of leukocytes disclosed fragmentation of deeply eosinophilic swollen collagen fibers. The surround ing cells appeared pyknotic and their differ entiation was not possible (fig. 3). When corneal sections were examined with ultraviolet illumination the following typical changes were noted (fig. 4). There was maximal fluorescence along the corneal line of most intense leukocyte concentration and collagen fragmentation. No fluorescence was evident beyond this line toward the lim bus except for autofluorescence of the gran ular leukocytes. The corneal tissue between the site of injection and the line showed dif fuse pale greenish fluorescence indicative of antigen diffusing from the site of injection toward the periphery of the cornea. No
ANAPHYLACTIC HYPERSENSITIVITY
Fig. 5 (Germuth et al.). Line of opacification appearing between the separate site of injection of antigen and antibody in a normal rabbit cornea. Three days after injection of 0.8 mg. of antibody and 0.5 mg. of fluorescein-tagged bovine serum albumin. blood vessels were found in the cornea. W h e n antigen and antibody were injected into separate sites in the normal cornea, a line of opacification appeared between the two sites of injection oriented at right angles to an imaginary line joining these (fig. 5 ) . After 24 to 48 hours, the line was histologically represented by an amorphous mass of deeply eosinophilic material which appeared to separate the collagen fibers and which traversed the full thickness of the cornea at right angles to the surface (fig. 6 ) . Leukocytic reaction at this stage was mild. This be came more prominent with time so that by the seventh day the lesion was similar to that seen among the sensitized animals 48 hours after intracorneal injection of antigen. F r a g mentation of the collagen fibers was not as evident in this experiment as it was in pre viously sensitized animals receiving intra corneal injection of antigen. W h e n the line of antigen-antibody precipi tation in the corneas of nonsensitized ani mals was followed grossly over a period of days following the simultaneous injection of antigen and antibody, it was observed that the line progressed as an opaque front away from the antigen side toward the antibody side. This was readily demonstable by mark ing the cornea at the initial line of precipita-
285
Fig. 6 (Germuth, et al.). Histologic section of the line shown in Figure 5. Twenty-four hours after injection of normal comae. The line is rep resented by an amorphous mass of deeply eosino philic material which appears to separate the colla gen fibers and which traverse the full thickness of the cornea at right angles to the surface. Leucocytic infiltration is mild at this stage. tion with India ink. Progressive development of the zone of opacification could be equated with antigen-antibody interaction as evi denced by gross examination under Wood's light and by fluorescence microscopy (fig. 7 ) . I t is believed that these experiments demonstate that antigen-antibody interaction can produce immediate damage to the connective tissue stroma of the avascular rabbit cornea and that this damage is exquisitely localized
Fig. 7 (Germuth, et al.). Fluorescence prepara tion of lesion similar to Figure 6. The intense white line represents a bright green line of fluores cence under ultraviolet light. Migration of the line as described in the text has resulted in a double line of fluorescence.
286
F. G. GERMUTH, JR. et al
at the site of interaction. Study of such le skin has obvious advantage not only for mor sions uncomplicated by the confusing effects phologic analysis but, more importantly, for and local participation of blood vessels as in chemical and enzymatic investigation. the hemorrhagic Arthus phenomenon in the The Johns Hopkins Hospital (5) REFERENCES
1. Hawn, C. van Z., and Janeway, C. A.: Histological and serological sequences in experimental hypersensitivity. J. Exper. Med., 85:571, 1947. 2. Germuth, F. G., Jr.: A comparative histologic and immunologic study in rabbits of induced hypersensitivity of the serum sickness type. J. Exper. Med., 97:257, 1953. 3. Germuth, F. G., Jr., Pace, M. G., and Tippett, J. C : The effect of sensitization to homologous and cross reactive antigen on the rate of antigen elimination and the development of allergic lesions. J. Exper. Med.. 101:135, 1955. 4. Sternberger, L. A., Maltaner, F., and DeWeerdt, J.: Estimation of circulating antibody-antibody complex. J. Exper. Med., 98:451,1953. 5. Weigle, W. O.i The nature of antigen-antibody complexes found in rabbits during an immune response to bovine serum albumin. J. Exper. Med., 107:653, 1958. 6. Mellors, R. C, Arias-Stella, J., Seigel, M., and Pressman, D.: Analytical pathology: II. Histopathologic demonstration of glomerular-localizing antibodies in experimental glomerulonephritis. Am. J. Path., 31:687, 1955. 7. Dixon, F. J., Vazquez, J. J., Weigle, W. O., and Cochrane, C. G.: Pathogenesis of serum sickness. Arch. Path., 65:18, 1958. 8. Germuth, F. G., Jr., and McKinnon, G. E.: Studies on the biological properties of antigen-antibody complexes: I. Anaphylactic shock induced by soluble antigen-antibody complexes in unsensitized normal guinea pigs. Bull. Johns Hopkins Hosp., 101:13, 1957. 9. Germuth, F. G., Jr.: Experimental anaphylactic glomerulonephritis and human nephritis in Proc. Ninth Annual Conf. on the Nephrotic Syndrome. Edited by Metcoff, J.: 1957, p. 39. 10. Schwab, L., Moll, F. C., Hall, T., Brean, H., Kirk, M., Hawn, C. van Z., and Janeway, C. A.: Effect of inhibition of antibody formation by x-radiation or nitrogen mustards on the histologic and serologic sequences, and on the behavior of serum complement, following single large injections of foreign protein. J. Exper. Med., 91:505, 1950. 11. Osier, A. G. Hawrisiak, M. M., Ovary, Z., Siqueira, M., and Bier, O. G.: Studies on the mech anism of hypersensitivity phenomena: II. The participation of complement in passive cutaneous anaphylaxis of the albino rat. J. Exper. Med., 106:811, 1957. 12. Cushman, W. F., Becker, E. L., and Wirtz, G.: Concerning the mechanism of complement actvie. III. Inhibitors of complement activity. J. Immunol., 79:80, 1957. 13. Ratnoff, G. O., and Lepow, I. H.: Some properties of an esterase derived from preparations of the first component of complement. J. Exper. Med., 106:327, 1957. 14. Rich, A. R., and Follis, R. H.: Studies on the site of sensitivity in the Arthus phenomenon Bull Johns Hopkins Hosp., 66:106, 1940.
DISCUSSION PHILLIPS
THYGESON
(San
Jose,
California):
The authors report recent work from their labora tory which indicates that connective tissue changes can be produced in the avascular cornea at the site of local antigen-antibody interaction. The changes noted were interpreted as degeneration or necrosis and were considered to be independent of leucocytic migration since similar changes were noted in ani mals rendered leucopenic by nitrogen mustard in jections. In each case they noted that connective tissue alteration occurred at the site of maximal precipitation of antigen-antibody complexes as dem onstrated by fluor-marking of the injected antigen. In view of the acute exudative inflammation, which was a characteristic feature of the experi ments, the elimination of leukocytic migration as a
factor in the necrotic reaction seems to be of spe cial importance. The development of grayish areas of opacification in the peripheral corneas of sensitized animals after injection of foreign protein into the center of the cornea immediately calls to mind the phenome non of marginal corneal infiltration and ring ulcer in staphylococcic blepharokeratoconjunctivitis in which staphylococciexotoxin is liberated in to the conjunctival sac in presumably sensitized animals. The resemblance to the precipitation ring that oc curs in the agar plate precipitin technique is also striking. The authors' demonstration of sharp lo calization of antigen in the ring of opacification is most convincing and their histologic demonstration of fragmentation of eosinophilic swollen collagen
ANAPHYLACTIC HYPERSENSITIVITY fibers leaves little doubt that damage occurred at the site of maximal antigen-antibody concentration. The authors' experiments in which antigen and antibody were injected into separate sites in the normal cornea also calls to mind the agar precipitin technique. The similarity of the damage to colla gen fibers at the line of opacification in the normal corneas as compared to the sensitized corneas is striking, and the delay in the development of this damage (seven days as compared to 48 hours) is understandable. Of special interest was the pro gression of the opacity away from the antigen site toward the antibody site and its relation to antigenantibody interaction as evidenced by fluorescence microscopy. This demonstration that antigen-antibody inter action can produce immediate damage to the con nective tissue stroma is of far-reaching importance. The advantage of an avascular structure such as the cornea for immunologic studies is obvious, and it seems likely that chemists and immunologists will turn to this tissue more and more in the fu ture. It is to be hoped that the authors will con tinue their important studies in this field. While they have well demonstrated the site of tissue in jury much more remains to determine the exact mechanism by which antigen-antibody-complement complexes produce tissue damage.
287
thorities feel that this syndrome is a manifestation of periarteritis nodosa. Good evidence is available to support the concept that the latter is a hyper sensitivity reaction. DR.
MICHAEL J. HOGAN
(San Francisco):
I
have seen a number of patients with identical le sions to the ring type of infiltrate which was seen in the initial slide, with many recurrences, each time with a complete ring infiltrate and each time a little greater distance into the cornea, the ring infiltrate always occurring just beyond the line of vascularization in the normal cornea itself. Also, I wonder if the authors have seen the arti cle in the last issue of the British Journal of Ophthalmology, in which identical lesions were pro duced by intracorneal and anterior chamber injec tions of heterologous sera. The one photograph shown in that article was of a typical ring infil trate in the peripheral cornea. DR. C. E. VAN ARNAM (closing): In answer to Dr. Hogan, I have not seen that article, but the na ture of the article and the references Dr. Falls and Dr. Thygeson have made to the resemblance of this experimentally produced ring to clinical cases prompts me to state that the original experiment was prompted because one of the authors, Dr. PrattJohnson actually observed clinically such a ring in filtrate in measles keratitis. It was one of the things he had been thinking about, and when the oppor DR. SEYMOUR P. HALBERT (New York): These observations, of course, are extremely intriguing. tunity was presented to explore it he was actually I would like to ask the authors whether there is a early in the work. possibility that the tissue effect that occurs in the The paper we have presented is in the nature of a cornea may be due merely to the physical presence preliminary report. Many of these things that have of the antigen-antibody precipitate, and has nothing been mentioned have also come to our minds. It to do with the secondary factors which are thought just seems that there is still a lot of work to be done to be involved in other hypersensitivity reactions. and that there are a lot of possibilities of using this One might possibly study this by using animals in particular technique. which the complement level has been decreased In reply to Dr. Halbert's question as to whether through some means or other. the physical presence of an antigen-antibody pre DR. HAROLD FALLS (Ann Arbor): Dr. Thygecipitate causes the damage, in contrast to the pos son has mentioned several clinical entities of hy sibility that we have suggested, that it is due to persensitivity etiology which exhibit corneal mani some biologic activity, I am sure we do not have an festation. These infiltrations are usually just inside answer. The senior author, Dr. Germuth, at the the limbus. I should like to add two additional present time is working on the problem of com clinical entities which have been postulated to have plement and its presence in these lesions. hypersensitivity causation and also exhibit corneal The next step in our work is to determine whether perilimbal infiltration: (1) Atopic eczema, reported complement was present in the antigen-antibody by Dr. Hogan and his associates; (2) Cogan's syn complex in the cornea. I am not able to report any drome—nonsyphilitic interstitial keratitis. Some au thing on that at this time.
G. GERMUTH, JR., M.D., L. B. SENTERFIT, M.D.,
A.
E. MAUMENEE, M.D.,
C. E. V A N ARNAM, M.D.,
J.
PRATT-JOHNSON, 1
AND A. D. POLLACK,
M.D., M.D.
Baltimore, Maryland
Previous work from this laboratory has been concerned primarily with the dynamics of antigen-antibody interaction and the rela tionship of this process to the tissue lesions in hypersensitivity of the serum sickness type. These investigations integrated with the work of others may be briefly sum marized. Studies by Hawn and Janeway 1 and our selves2' 3 have demonstrated that the lesions of serum sickness appear as an animal be gins to form antibody while antigen is still present in the circulation and tissue fluids. With continued production of antibody, an tigen is eliminated, free antibody appears in the circulation, and the organic manifesta tions of serum sickness resolve. Since anti gen is present in the blood during the early antibody response, it was suggested by us some years ago 2 - 3 that the tissue lesions might arise secondary to the transport of an tibody by antigen in the form of soluble an tigen-antibody complexes. In the intervening years, Sternberger, et al.,4 and Weigle5 have demonstrated antigen-antibody complexes in the circulation. Mellors, et al.,6 and Dixon, et al.,7 employing fluorescent antigen and anti bodies found antigen and gamma globulin lo calized in the tissue lesions of hypersensitiv ity. It is not known whether the localization of gamma globulin in the tissue represents specifically fixed antibody. Other work from our laboratory has indicated that soluble anti gen-antibody complexes produced in vitro may elicit anaphylactic shock in guinea pigs, produce Arthus reactions in the skin, and in* From the Department of Pathology and the Department of Ophthalmology, The Johns Hop kins University Medical School. t Fight-for-Sight fellow of the National Council to Combat Blindness, New York, 19S7-S8. 282
duce glomerular and splenic lesions of the serum sickness type following infusion into normal rabbits. 8 ' 9 Presumably these reactions require complement. 10 '" There is then consid erable evidence that soluble antigen-antibody complexes exhibit significant biologic activ ity and that they may play a leading role in the development of allergic reactions. It is clear that important strides have been made in an area which may be called the immunogenesis of immediate-type allergic re actions. Two of the most important problems which still demand clarification concern firstly, the mechanism by which antigen-an tibody-complement complexes injure tissue, and, secondly, the site of tissue injury. In regard to the precise biochemical mech anism through which antigen-antibody-com plement interaction results in tissue injury little is known. Studies currently being car ried on by others 12 ' 13 on the role of comple ment in the hemolysis of red blood cells could provide data which might contribute to an un derstanding of tissue damage in allergic reactions. Many workers believe that comple ment is concerned in the activation of an enzymatic system in the presence of reactive antigen and antibody. 12 ' 13 In regard to the site of tissue injury in al lergic reactions, numerous studies have been made. There seems little doubt that anaphy lactic reactions are manifested in the vascu lar and connective tissue systems. Whether damage to the vascular system is primarily an expression of alteration in the connective tissue which is indissolubly associated with blood vessels or whether these changes are due to endothelial injury has never been re solved. Since marked alteration of the con nective tissues is prominent in hypersensitiv ity, the first of these possibilities seems more
ANAPHYLACTIC HYPERSENSITIVITY
acceptable and warrants further investiga tion. Some years ago, Rich and Follis" failed to produce a typical hemorrhagic Arthus re action in the rabbit cornea unless vascularization had been previously induced. They noted "slight edema, slight polymorphonuclear infiltration, and moderate swelling of the corneal fibers" 24 and 48 hours after in jection of horse serum into the avascular cornea of sensitized animals. It would seem that these workers produced a true Arthus phenomenon in the vascularized corneas of sensitized animals. However, this might be expected under the conditions of the experi ment since vascularization of the cornea in effect converts the cornea into a structure very similar to skin. Since the cornea consists of almost pure connective tissue comprised of fibroblasts, collagen, and reticulum fibers, and amor phous "ground substance" far removed from a blood supply, and since there is so much experimental and pathologic evidence of con nective tissue damage in hypersensitivity, it was deemed useful to employ this tissue in further studies of hypersensitivity, using quantitative immunologic methods to see whether connective tissue damage could in deed be related to antigen-antibody interac tion in the absence of blood vessels. Recent work in our laboratory has disclosed that connective tissue change can be produced in the avascular cornea at the site of local anti gen-antibody interaction. These experiments were of two designs. In one type, appropri ate quantities of bovine serum albumin or bovine serum gamma globulin were injected into the avascular cornea of passively or ac tively sensitized rabbits. In the second type of experiment, bovine serum albumin and its corresponding antibody were simultaneously injected into different sites of the avascular corneas of normal rabbits. In all experi ments, alterations in the connective tissue were observed which have been interpreted as those of degeneration or necrosis. Al though these changes were accompanied by
283
acute exudative inflammation, they did not appear to be dependent on leucocyte migra tion since similar changes were noted in ani mals which had been rendered leucopenic by nitrogen mustard injections. In each case, the connective tissue alteration occurred at the site of maximal precipitation of antigenantibody complexes as demonstrated by fluor-marking of the injected antigen. Following the injection of 0.1 ml. of fluor-labeled foreign protein into the center of the cornea of actively or passively sensi tized animals, the limbus appeared grossly inflammed six to eight hours later. From 12 to 24 hours later, a grayish arc of opacification appeared in the periphery of the cornea. It was separated from the limbus by a clear area one to three mm. in width (fig. 1). Further observation for an additional 24 hours generally disclosed completion of the arc to form a concentric ring of opacification. Examination of such eyes under Wood's light in the living animal revealed
Fig. 1 (Germuth, et al.). Living eye in rabbit previously sensitized to bovine albumin. Twentyfour hours after injection of 1.0 mg. fluor-bovine albumin into center of cornea. Note opaque ring in periphery of cornea with clear cornea between ring and limbus. Dilatation and engorgement of limbal and bulbar conjunctiva! vessels with petechial hemorrhages. Under Wood's light the gray opaque ring presented bright green fluorescence. (The two bright spots are highlights.)
2M
F. G. GERMUTH, JR. et al
Fig. 2 (Germuth, et al.). Transverse section of peripheral portion of cornea and adjacent limbus. Rabbit previously sensitized with bovine albumin. Received injection of 1.0 mg. fluor-bovine serum albumin in center of cornea 30 hours before death. Hemorrhage and neutrophilic leukocytic infiltra tion around limbal vascular loops. Sharp transverse line of leukocytic infiltration and collagen degenera tion distant from limbus. This line corresponds to a section through the opaque ring seen grossly. Note leukocytes migrating from limbus across clear corneal zone to line. No blood vessels are present in cornea.
sharp localization of antigen in the ring of opacification. Histologic examination of corneas show ing such rings (fig. 2) revealed a sharp line of concentrated leukocytes traversing the cornea from Bowman's to Descement's mem brane. The leukocytes had evidently mi grated from the limbal side as evidenced
Fig. 3 (Germuth et al.). Moderate magnification of cornea to illustrate the histologic appearance of the opaque ring. Leukocytic infiltration and swell ing and degeneration of corneal stroma.
Fig. 4 (Germuth et al.). Fluorescence prepara tion of lesion similar to that of Figure 3. The in tense white line represents a bright green line of fluorescence under ultraviolet illumination due to fixation of fluor-antigen by antibody diffusing from limbus in a sensitized animal 30 hours after intracorneal injection 1.0 mg. fluor-bovine serum albu min. The line of fluorescence coincides precisely with the line of leucocyte infiltration and collagen degeneration. (The bright appearance of the cells is due to autofluorescence.)
by the presence of scattered leukocytes be tween the limbus and the sharp line of leu kocytic infiltration. The limbal vessels were dilated and engorged and the loose connec tive tissue around them was infiltrated by leukocytes, red blood cells, and fibrin. Closer examination of the corneal line of leukocytes disclosed fragmentation of deeply eosinophilic swollen collagen fibers. The surround ing cells appeared pyknotic and their differ entiation was not possible (fig. 3). When corneal sections were examined with ultraviolet illumination the following typical changes were noted (fig. 4). There was maximal fluorescence along the corneal line of most intense leukocyte concentration and collagen fragmentation. No fluorescence was evident beyond this line toward the lim bus except for autofluorescence of the gran ular leukocytes. The corneal tissue between the site of injection and the line showed dif fuse pale greenish fluorescence indicative of antigen diffusing from the site of injection toward the periphery of the cornea. No
ANAPHYLACTIC HYPERSENSITIVITY
Fig. 5 (Germuth et al.). Line of opacification appearing between the separate site of injection of antigen and antibody in a normal rabbit cornea. Three days after injection of 0.8 mg. of antibody and 0.5 mg. of fluorescein-tagged bovine serum albumin. blood vessels were found in the cornea. W h e n antigen and antibody were injected into separate sites in the normal cornea, a line of opacification appeared between the two sites of injection oriented at right angles to an imaginary line joining these (fig. 5 ) . After 24 to 48 hours, the line was histologically represented by an amorphous mass of deeply eosinophilic material which appeared to separate the collagen fibers and which traversed the full thickness of the cornea at right angles to the surface (fig. 6 ) . Leukocytic reaction at this stage was mild. This be came more prominent with time so that by the seventh day the lesion was similar to that seen among the sensitized animals 48 hours after intracorneal injection of antigen. F r a g mentation of the collagen fibers was not as evident in this experiment as it was in pre viously sensitized animals receiving intra corneal injection of antigen. W h e n the line of antigen-antibody precipi tation in the corneas of nonsensitized ani mals was followed grossly over a period of days following the simultaneous injection of antigen and antibody, it was observed that the line progressed as an opaque front away from the antigen side toward the antibody side. This was readily demonstable by mark ing the cornea at the initial line of precipita-
285
Fig. 6 (Germuth, et al.). Histologic section of the line shown in Figure 5. Twenty-four hours after injection of normal comae. The line is rep resented by an amorphous mass of deeply eosino philic material which appears to separate the colla gen fibers and which traverse the full thickness of the cornea at right angles to the surface. Leucocytic infiltration is mild at this stage. tion with India ink. Progressive development of the zone of opacification could be equated with antigen-antibody interaction as evi denced by gross examination under Wood's light and by fluorescence microscopy (fig. 7 ) . I t is believed that these experiments demonstate that antigen-antibody interaction can produce immediate damage to the connective tissue stroma of the avascular rabbit cornea and that this damage is exquisitely localized
Fig. 7 (Germuth, et al.). Fluorescence prepara tion of lesion similar to Figure 6. The intense white line represents a bright green line of fluores cence under ultraviolet light. Migration of the line as described in the text has resulted in a double line of fluorescence.
286
F. G. GERMUTH, JR. et al
at the site of interaction. Study of such le skin has obvious advantage not only for mor sions uncomplicated by the confusing effects phologic analysis but, more importantly, for and local participation of blood vessels as in chemical and enzymatic investigation. the hemorrhagic Arthus phenomenon in the The Johns Hopkins Hospital (5) REFERENCES
1. Hawn, C. van Z., and Janeway, C. A.: Histological and serological sequences in experimental hypersensitivity. J. Exper. Med., 85:571, 1947. 2. Germuth, F. G., Jr.: A comparative histologic and immunologic study in rabbits of induced hypersensitivity of the serum sickness type. J. Exper. Med., 97:257, 1953. 3. Germuth, F. G., Jr., Pace, M. G., and Tippett, J. C : The effect of sensitization to homologous and cross reactive antigen on the rate of antigen elimination and the development of allergic lesions. J. Exper. Med.. 101:135, 1955. 4. Sternberger, L. A., Maltaner, F., and DeWeerdt, J.: Estimation of circulating antibody-antibody complex. J. Exper. Med., 98:451,1953. 5. Weigle, W. O.i The nature of antigen-antibody complexes found in rabbits during an immune response to bovine serum albumin. J. Exper. Med., 107:653, 1958. 6. Mellors, R. C, Arias-Stella, J., Seigel, M., and Pressman, D.: Analytical pathology: II. Histopathologic demonstration of glomerular-localizing antibodies in experimental glomerulonephritis. Am. J. Path., 31:687, 1955. 7. Dixon, F. J., Vazquez, J. J., Weigle, W. O., and Cochrane, C. G.: Pathogenesis of serum sickness. Arch. Path., 65:18, 1958. 8. Germuth, F. G., Jr., and McKinnon, G. E.: Studies on the biological properties of antigen-antibody complexes: I. Anaphylactic shock induced by soluble antigen-antibody complexes in unsensitized normal guinea pigs. Bull. Johns Hopkins Hosp., 101:13, 1957. 9. Germuth, F. G., Jr.: Experimental anaphylactic glomerulonephritis and human nephritis in Proc. Ninth Annual Conf. on the Nephrotic Syndrome. Edited by Metcoff, J.: 1957, p. 39. 10. Schwab, L., Moll, F. C., Hall, T., Brean, H., Kirk, M., Hawn, C. van Z., and Janeway, C. A.: Effect of inhibition of antibody formation by x-radiation or nitrogen mustards on the histologic and serologic sequences, and on the behavior of serum complement, following single large injections of foreign protein. J. Exper. Med., 91:505, 1950. 11. Osier, A. G. Hawrisiak, M. M., Ovary, Z., Siqueira, M., and Bier, O. G.: Studies on the mech anism of hypersensitivity phenomena: II. The participation of complement in passive cutaneous anaphylaxis of the albino rat. J. Exper. Med., 106:811, 1957. 12. Cushman, W. F., Becker, E. L., and Wirtz, G.: Concerning the mechanism of complement actvie. III. Inhibitors of complement activity. J. Immunol., 79:80, 1957. 13. Ratnoff, G. O., and Lepow, I. H.: Some properties of an esterase derived from preparations of the first component of complement. J. Exper. Med., 106:327, 1957. 14. Rich, A. R., and Follis, R. H.: Studies on the site of sensitivity in the Arthus phenomenon Bull Johns Hopkins Hosp., 66:106, 1940.
DISCUSSION PHILLIPS
THYGESON
(San
Jose,
California):
The authors report recent work from their labora tory which indicates that connective tissue changes can be produced in the avascular cornea at the site of local antigen-antibody interaction. The changes noted were interpreted as degeneration or necrosis and were considered to be independent of leucocytic migration since similar changes were noted in ani mals rendered leucopenic by nitrogen mustard in jections. In each case they noted that connective tissue alteration occurred at the site of maximal precipitation of antigen-antibody complexes as dem onstrated by fluor-marking of the injected antigen. In view of the acute exudative inflammation, which was a characteristic feature of the experi ments, the elimination of leukocytic migration as a
factor in the necrotic reaction seems to be of spe cial importance. The development of grayish areas of opacification in the peripheral corneas of sensitized animals after injection of foreign protein into the center of the cornea immediately calls to mind the phenome non of marginal corneal infiltration and ring ulcer in staphylococcic blepharokeratoconjunctivitis in which staphylococciexotoxin is liberated in to the conjunctival sac in presumably sensitized animals. The resemblance to the precipitation ring that oc curs in the agar plate precipitin technique is also striking. The authors' demonstration of sharp lo calization of antigen in the ring of opacification is most convincing and their histologic demonstration of fragmentation of eosinophilic swollen collagen
ANAPHYLACTIC HYPERSENSITIVITY fibers leaves little doubt that damage occurred at the site of maximal antigen-antibody concentration. The authors' experiments in which antigen and antibody were injected into separate sites in the normal cornea also calls to mind the agar precipitin technique. The similarity of the damage to colla gen fibers at the line of opacification in the normal corneas as compared to the sensitized corneas is striking, and the delay in the development of this damage (seven days as compared to 48 hours) is understandable. Of special interest was the pro gression of the opacity away from the antigen site toward the antibody site and its relation to antigenantibody interaction as evidenced by fluorescence microscopy. This demonstration that antigen-antibody inter action can produce immediate damage to the con nective tissue stroma is of far-reaching importance. The advantage of an avascular structure such as the cornea for immunologic studies is obvious, and it seems likely that chemists and immunologists will turn to this tissue more and more in the fu ture. It is to be hoped that the authors will con tinue their important studies in this field. While they have well demonstrated the site of tissue in jury much more remains to determine the exact mechanism by which antigen-antibody-complement complexes produce tissue damage.
287
thorities feel that this syndrome is a manifestation of periarteritis nodosa. Good evidence is available to support the concept that the latter is a hyper sensitivity reaction. DR.
MICHAEL J. HOGAN
(San Francisco):
I
have seen a number of patients with identical le sions to the ring type of infiltrate which was seen in the initial slide, with many recurrences, each time with a complete ring infiltrate and each time a little greater distance into the cornea, the ring infiltrate always occurring just beyond the line of vascularization in the normal cornea itself. Also, I wonder if the authors have seen the arti cle in the last issue of the British Journal of Ophthalmology, in which identical lesions were pro duced by intracorneal and anterior chamber injec tions of heterologous sera. The one photograph shown in that article was of a typical ring infil trate in the peripheral cornea. DR. C. E. VAN ARNAM (closing): In answer to Dr. Hogan, I have not seen that article, but the na ture of the article and the references Dr. Falls and Dr. Thygeson have made to the resemblance of this experimentally produced ring to clinical cases prompts me to state that the original experiment was prompted because one of the authors, Dr. PrattJohnson actually observed clinically such a ring in filtrate in measles keratitis. It was one of the things he had been thinking about, and when the oppor DR. SEYMOUR P. HALBERT (New York): These observations, of course, are extremely intriguing. tunity was presented to explore it he was actually I would like to ask the authors whether there is a early in the work. possibility that the tissue effect that occurs in the The paper we have presented is in the nature of a cornea may be due merely to the physical presence preliminary report. Many of these things that have of the antigen-antibody precipitate, and has nothing been mentioned have also come to our minds. It to do with the secondary factors which are thought just seems that there is still a lot of work to be done to be involved in other hypersensitivity reactions. and that there are a lot of possibilities of using this One might possibly study this by using animals in particular technique. which the complement level has been decreased In reply to Dr. Halbert's question as to whether through some means or other. the physical presence of an antigen-antibody pre DR. HAROLD FALLS (Ann Arbor): Dr. Thygecipitate causes the damage, in contrast to the pos son has mentioned several clinical entities of hy sibility that we have suggested, that it is due to persensitivity etiology which exhibit corneal mani some biologic activity, I am sure we do not have an festation. These infiltrations are usually just inside answer. The senior author, Dr. Germuth, at the the limbus. I should like to add two additional present time is working on the problem of com clinical entities which have been postulated to have plement and its presence in these lesions. hypersensitivity causation and also exhibit corneal The next step in our work is to determine whether perilimbal infiltration: (1) Atopic eczema, reported complement was present in the antigen-antibody by Dr. Hogan and his associates; (2) Cogan's syn complex in the cornea. I am not able to report any drome—nonsyphilitic interstitial keratitis. Some au thing on that at this time.