Ultrastructural evidence for lack of tissue damage in a local immune complex reaction

Virchows Arch. B Cell Path. 18, 213--224 (1975) �9 by Springer-Verlag 1975

Ultrastructural Evidence for Lack of Tissue Damage in a Local Immune Complex Reaction A S t u d y of a Mild Passive Ar thus Reac t ion*

Klaus Weber, Hiroaki Ueki **, He lmn t H. Wolff, and Otto Braun-Falco

Dermatologische Klinik und Poliklinik der Universitiit Miinchen (Direktor: Prof. Dr. O. Braun-Falco)

Received March 24,1975

Summary. An electron microscopic study of a mild reversed passive Arthus reaction (RPAR) was performed using a horseradish peroxidase (HRP)-anti-HRP system to demon- strate the biological usefulness of the process which exists to clear tissue of immune complexes.

To disclose the antigen, HRP, the biopsy material was subjected to a peroxidase reaction. HRP was mainly detected within irregular electron-dense precipitates which were considered insoluble HRP-anti-HRP immune complexes.

Neutrophils were found to phagocytose and digest the deposited immune complexes in a similar way as described previously. But there was no vascular and other tissue damage.

This study provides morphological evidence that the process of clearing tissue of immune complexes needs not be harmful to the host.

Key words: Arthus Reaction - - Electron Microscopy - - Immune Complexes Horse- radish Peroxidase - - Ultrastructure.

Many exper imental invest igat ions using Arthus reactions as models have been

presented to show the deleterious effects exerted by the process of the removal

from the tissue of immune complexes (Cochrane, 1965; Letterer, 1967; Cochrane,

1968). The quan t i t a t ive evaluat ion of Ar thus reactions has, however, shown tha t

clinical signs of tissue damage may be absent in mild Arthus reactions (Culbertson, 1935; Fischel and Kaba t , 1947; Cochrane, 1965; Fischel, 1967). I t was the aim of the present s tudy to provide morphological evidence based on electron microscopical f indings showing tha t a process exists which can clear tissue from immune complexes wi thout necessarily result ing in dest ruct ion of tha t tissue. We thus in tended to demonst ra te the biological usefulness of this clearing process.

There have only been a few ultrastructural studies on the passive Arthus phenomenon so far (Daems and Oort, 1962; Fernando and Movat, 1963; Uriuhara and Movat, 1966). Daems and Oort investigated the process of phagocytosis of immune complexes during the course of a reversed passive Arthus reaction (RPAR). Fernando and Movat demonstrated the deposition of ferritin-anti-ferritin complexes in blood vessels during the first 30 rain of an RPAR. Uriuhara and Movat performed an extensive study of the events occuring between 30 min and 24 h after induction of an RPAR. In the latter investigation, pronounced vascular changes, such as homogenisation of endothelial cells and vascular necrosis, were observed.

In the mentioned studies, bovine serum albumin (BSA) and ferritin were used as antigens. Recently, horseradish peroxidase (HRP) has been used as antigen in light (Straus, 1972) and electron microscopic (Venkatachalam and Cotran, 1970; Graham and Griffin, 1972) studies on the active Arthus reaction. We, too, applied HRP in the present study on the passive

* Supported by the Deutsche Forschungsgemeinschaft. ** Fellow of the Alexander von Humboldt-Stiftung; present address: Dpt. of Dermatology, Kawasaki Medical College, Matsushima 577, 701-01 Kurashiki-shi, Japan.

16 Virchows Arch. B Cell Path., Vol. 18

214 K. Weber et al.

Arthus reaction because H R P has a low molecular weight and is readily identified after the performance of a reliable enzyme-histochemical reaction. H R P is known to retain enough enzyme activity for histoehemical demonstration after it has reacted with specific precipitating anti-i-IRP antibodies (Leduc et al., 1968).

Our previous light (Ueki et al., 1974) and limited ultrastructural (Weber et al., 1974) in- vestigations on mild RPAWs did not conclusively prove the purpose which is the subject of the present study. I t was thus necessary to perform an electron microscopical investigation of the entire course of a mild RPAR to reach a final conclusion.

Materials and Methods Antigen. H R P (RZ value 1.0_~90 units/mg) purchased from Schuchardt, Munich, was

used as antigen. Shortly before usage, it was dissolved in phosphate buffered saline (PBS). When the antigen was tested against the antiserum using immuno-electrophoresis it was found to consist of 4 different fractions to which the sensitized rabbits had produced antibodies (see Ueki et al., 1974).

Antiserum. Antiserum containing precipitating ant i -HRP antibodies was obtained from several albino rabbits as described in a previous paper (Ueki et al., 1974). A serum lot was only denoted antiserum when it yielded precipitation arcs with H R P in dilutions of up to 1:8 or 1:16 as checked by the double diffusion technique of Ouchterlony.

Animals. Several non-sensitized albino rabbits weighing 4-5 kg, obtained from a local dealer, were used to elicit the RPAR.

Introduction o/the RPAR. Each rabbit received 0.1 ml antiserum intracutaneously into each of 3 sites of the ears' skin. 10-15 min later, 20 mg H R P were injected intravenously into each rabbit.

Biopsies. In ech rabbit biopsies were taken from each of the induced R P A R lesions at the following times after injection of the H R P which served as starting point of the RPAR : 5 min and 15 min, 1 h, 4 h, 24 h and 48 h; one of the 3 RPAR lesions was biopsied as late as 24 h or 48 h after induction of the R P A R to get a chance for macroscopical evaluation of the lesions in each animal.

Preparation of Tissue. The biopsy material was immediately minced into small pieces and fixed in a mixture containing 2% formaldehyde, 2.5% glutaraldehyde and 0.5% CaC12 in 0.1 M cacodylate buffer (pH 7.5) for 90 min at 4~ cut in the same fixative for 30 min, washed in 0.1 M cacodylate buffer containing 7.5% sucrose (pH 7.5) overnight at 4~ subjected to the peroxidase reaction of Graham and Karnovsky (1966) for 1 h at room temperature, washed again in the above mentioned cacodylate buffer, post-fixed in 1% OsO 4 in 0.1 M cacodylate buifer (pH 7.5) at 4~ washed three times in veronal acetate buffer (pH 5.0) at room temperature, treated in 0.5% uranyl acetate in veronal acetate buffer (pH 5.0) for 2 h, dehydrated in alcohol and propylene oxide and embedded in epon.

Peroxidase Reaction. The method of Graham and Karnovsky (1966) was used; a few biopsy pieces were treated without H202 for control purposes.

Light Microscopy. Semithin (1 iz) sections were cut for light microscopy, left unstained or were stained with methylene blue-Azur II.

Electron Microscopy. Thin sections were cut with diamond knives. The sections were contrasted with uranyl acetate and lead citrate. Electron microscope: Zeiss EM 9A.

Controls. 3 kinds of controls were performed in non-sensitized rabbits: injection of 20 mg H R P intravenously and normal rabbit serum intracutaneously (3 sites); 3 injections of anti- sermn without the subsequent administration of H R P ; and, in 1 rabbit, 20 mg t I R P were injected intravenously without the administration of antiserum or normal rabbit serum. The time sequence chosen for the biopsies of the controls were the same as in the main trial, but did not extend 6 h.

Results 1. Macroscopic Appearance o/ the Arthus Lesion~

T h e mac roscop ica l changes of t h e sk in si tes in wh ich t h e R P A R was i n d u c e d

( R P A R lesions) cons i s t ed of s l ight e d e m a and redness in an a rea 1-2 cm in dia-

m e t e r a t t h e h e i g h t of t h e A r t h u s reac t ion . N o h e m o r r h a g e or necrosis were no t ed .

Passive Arthus Reaction--Lack of Tissue Damage 215

2. Light Microscopy a) General Remarks. No morphological abnormali ty of the blood vessels was

noted throughout the entire Arthus reaction. The lymph vessels were sometimes greatly distended and did not contain any reaction product within their lumina. A few erythrocytes were found perivascularly.

b) Findings 5 rain niter Induction o/the RPAR. Many small blood vessels, most of which were venules, showed a heavy deposition of granular reaction product within their walls and to a lesser extent in their immediate vicinity and lumina. In addition, quite a number of neutrophils were seen within and outside the affected blood vessels. The neutrophils contained a lot of peroxidase-positive granules and an occasional vacuole. Only a few capillaries were affected.

c) Findings after 15 rain and I h. They were basically similar to the results observed after 5 min, but the reaction was more intense. Some more blood vessels including some more capillaries were affected. More vacuoles appeared within ueutrophils.

d) Findings alter 4 h. Fewer blood vessels were affected than after 1 h. Granular reaction product was detected within the walls of some blood vessels which were not infiltrated by neutrophils. Quite a lot of granular reaction product could sometimes be seen in the vicinity of the venules.

e) Findings alter 24 h and 48 h. Only an occasional blood vessel wall contained reaction product 24 and 48 h after induction of the RPAR. Besides a moderate and often perivascular infiltrate of neutrophils, quite a number of mononuclear cells were seen many of which contained peroxidase-positive granules and/or parts of neutrophils within their cytoplasm.

3. Electron Microscopy a) General Remarks. The electron microscopical findings corresponded to the

light microscopical results. The reaction product consisted in almost all instances of irregular electron-dense precipitates which were 0.2-2.0 ~m in diameter (Figs. 1--5). These precipitates had deposited within the blood vessel wall outside of vascular cells and to a lesser extent in the vicinity and within the lumina of the affected blood vessels. The precipitates were frequently seen on, or near, collagen fibrils and the vascular basement membrane. Sometimes, precipitates were not attached to any structure. The plasma membranes of endothelial cells, pericytes, neutrophils and macrophages and the inner leaflet of phagocytic vacuoles of neutrophils were blackened when the precipitates were present in their vicinity (Figs. 1--5).

On rare occasions, a more diffuse and less electron-dense kind of reaction product was seen within the lumina of blood vessels and in the intercellular space of adjacent endothelial cells. Structures near this type of reaction product and near erythroeytes were not blackened.

There was no sign of damage to blood vessels, collagen fibrils or connective tissue cells (Figs. 1--5) but there was some evidence of mild vacuolization within occasional endothelial cells (Figs. 4--5). The basement membrane of the blood vessels was not altered unless it was lacking or fragmented at sites where in- f lammatory cells (mostly neutrophils) were paving their way through the blood

16"

216 K. Weber et al.

Fig. 1. Blood vessel 5 min after induction of the RPAR. Several HRP-anti-HRP immune complexes (arrows) have deposited within and just outside the blood vessel. A few complexes

are phagoeytosed by neutrophils (N). E endothelial cell; L lumen; P pericyte. • 8000


Fig. 2. HRP-anti-HRP immune complexes (arrows) outside, within the cytoplasm and within 2 vacuoles of a neutrophil which is embraced by another cell 5 min after induction of the

RPAR. • 14400

vessel wall (Figs. l, 3--5). There was no evidence of platelet aggregation and/or thrombosis. A few erythrocytes had escaped the intravascular lumina. Lymph vessels were sometimes greatly distended. Degranulation of mast cells and gaps between endothelial cells were lacking.

b) Findings a/ter 5 rain. There was a rather heavy deposition of the irregular electron-dense precipitates within the wails, and to a lesser extent outside and within the lumina, of many of the blood vessels most of which were venules (Fig. 1). Some precipitates were already seen within the cytoplasm of neutrophils which were found in considerable numbers within the lumina, the walls and the immediate vicinity of the affected blood vessels (Figs. 1 and 2). Many of the neutrophils within which the electron-dense precipitates were found still contained most of their granules (Fig. 2).

c) Findings alter 15 rain and I h. The deposition of the electron-dense precipitates was more pronounced and more neutrophils were seen to be involved in phagocytosing these precipitates (Figs. 3--5). Many neutrophils had lost some or most of their granules and contained one to several large vacuoles (Figs. 3--5).

218 K. Weber et al.

Fig. 3. A neutrophil (N) and an eosinophil (Eo) emigrate from the lumen (L) of a blood vessel 15 min after induction of the RPAR. HRP-anti-HRP immune complexes are present within

or near the neutrophi]. • 16200

Sometimes, the precipitates were less electron-dense within than outside the phagocytotic vacuoles. These vacuoles replaced almost the entire cytoplasm of several neutrophils some of which were embraced by mononuclear cells. In one instance, an eosinophil was seen to have phagocytosed several electron-dense precipitates (Fig. 4).

d) Findings alter 4 h. The deposition of the electron-dense precipitates was less pronounced. A few small blood vessels contained precipitates without being infiltrated by inflammatory ceils. Many capillaries were devoid of precipitates. Desintegration of neutrophils and phagocytosis of neutrophils by macrophages were denoted.

e) Findings alter 24 h and 48 h. All blood vessels examined were free of the electron-dense precipitates. However, precipitates were still present in a few areas of the interstitial tissue 48 h after induction of the RPAI~. The interstitial tissue was infiltrated by a moderate number of mononuclear cells and neutrophils most of which were attached to or ingested by macrophages.

Controls. The electron-dense irregular precipitates observed in the main trial were not seen in any of the control sections. We could, however, find the less electron-dense material within the lumina of small blood vessels, within pino- cytotic vesicles and in the space between adjacent endothelial cells in biopsy material taken 5 min to 6 h after the intravenous injection of H R P regardless


Fig. 4. Blood vessel 15 min after induction of the RPAR. Many HRP-anti-HRP immune complexes (arrows), several neutrophils (N) and one eosinophil (Eo) can be seen within the blood vessel walt. The eosinophil has taken up a few complexes. The immune complexes, but not the erythrocytes (double arrows) show the adherence phenomenon. E endothelial cell;

L lumen; P pericyte. • 6500

Fig. 5. Small blood vessel 1 h after induction of the RPAR. Several tlRP-anti-I-I:RP immune complexes (arrows) are present within the wall or outside the blood vessel and within or near neutrophils (N). Note t ha t the immune complexes within the phagocytic vacuoles of neutrophils (V) are less electron-dense than the complexes outside the vacuoles. The vascular cells

and the vascular basement membrane are intact. E endothelial cell; P pericyte. • 20000


whether or not normal rabbit serum had been administered intracutaneously. The less electron-dense reaction product was not detected in biopsy material taken from rabbits in which no H R P was injected.

Blackening of cell membranes adjacent to exogenous t IRP was absent. Black- ening of structures near erythrocytes was sometimes noted in poorly preserved biopsy material.

Discussion

The present study demonstrates that the removal of immune complexes from the tissue does not necessarily lead to damage of that tissue. This result was derived from an electron-microscopical investigation of the course of a mild RPAR using H R P as antigen.

The kind of H R P chosen by us as antigen was felt to fulfill the purpose of this study although it consisted of at least 4 different fractions as tested by immuno- electrophoresis. The moderate dosage of 20 mg H R P was a compromise arrived at to minimize the non-specific background, which would appear if higher doses were applied, and to create a sufficient amount of immune complexes. Furthermore, 20 mg H R P injected into a control rabbit was found to be still high enough a dosage to see H R P escape through the walls of blood vessels in a similar fashion as shown by Karnovsky (1967). The way in which antigen and antiserum were injected ascertained the desired mildness of the RPAR and the formation of a sufficient number of insoluble immune complexes.

To create comparable conditions in the RPAI~ lesions, antiserum was injected 10-15 min prior to the administration of the antigen to allow the antiserum to spread. The injection of antiserum induced the known (Oort and van Rijssel, 1961; Cochrane, 1968) non-specific accumulation of neutrophils in the injection sites.

A prerequisite for the evaluation of the induced immune complex reaction was the safe identification of insoluble HRP-ant i -HRP immune complexes. In fact, we found it easy to distinguish between 2 kinds of electron-dense deposits: the more diffuse, less electron-dense deposit was interpreted as representing free, non-complexed H R P and the irregular, strongly electron-dense deposit as indicat- ing H R P bound to precipitating ant i-HRP antibodies within precipitated immune complexes. Even within neutrophils, HRP-ant i -HRP immune complexes could in most instances be surprisingly well distinguished from the peroxidase-positive granules. The partial loss of electron-density of HRP-ant i -HRP immune complexes within neutrophilic vacuoles was probably due to their being digested by lysosomal enzymes. Our interpretation regarding the identification of the immune complexes was strongly supported by similar or identical ultrastructural findings of other authors (Venkatachalam and Cotran, 1970; Graham and Griffin, 1972; Steinman and Cohn, 1972) and by our own experience (Weber et al., 1973; Ueki et al., 1974; Weber st al., 1974). Moreover, in a light microscopic study, Straus (1972) was able to identify both antigen and antibody within HRP-ant i -HRP immune complexes.

The suggestion that immune complexes are present in (Oort and van Rijssel, 1961) or are phagocytosed by (Graham and Griffin, 1972) endothelial cells could

222 K. Weber et al.

not be supported by our findings; Venkatachalam and Cotran (1970) are in agreement with us on this point.

The blackening of cell membranes in the vicinity of HRP-ant i -HRP immune complexes may be due to a diffusion artifact (Novikoff et al., 1972; Seligman et al., 1973; Fahimi, 1973) or a specific attachment phenomenon. We favour the assumption that our observation represents a specific process; it may be the counterpart of what is known as immune adherence (Nelson, 1963; Cochrane, 1968) and/or it may represent complement-independent attachment of immune complexes to receptors on the cell surface similar to the findings obtained by Steinman and Cohn (1972).

The blackening of membranes of phagocytotic neutrophilic vacuoles containing immune complexes may be interpreted along the same lines as indication of what has been called immune phagocytosis.

Ample evidence has been presented to show that neutrophils cause tissue damage during the course of an Arthus reaction (Stetson, 1951 ; Humphrey, 1955; Cochrane and Weigle, 1958; Cochrane et al., 1959; Parish, 1969). In our biopsy material, neutrophils rapidly accumulated, phagocytosed and digested the HRP- ant i -HRP immune complexes, and finally they decayed. Previous electron- microscopic studies on various types of Arthus reactions have led to similar findings (Daems and Oort, 1962; Sabesin and Banfield, 1963; Fernando and Movat, 1963; Gieseking, 1966; Uriuhara and Movat, 1966; Grant etal . , 1967; Venkatachalam and Cotran, 1970; Graham and Griffin, 1972; Weber et al., 1974). But in contrast to other studies on the Arthus reaction our results show that the examined tissue was essentially not demaged. Furthermore, platelet aggregation and thrombosis did not occur which parallels the findings of Uriuhara and Movat (1966). Moreover, our observations reveal that damage to the vascular basement membrane is not a prerequisite for the development of an Arthus reaction, although it has been shown by Cochrane and Aikin (1966) that disruption of the vascular basement membrane may occur in an RPAR and in a reaction induced by locally injected antibody to vascular basement membrane due to the action of substances released by neutrophils.

In conclusion, deposition of insoluble immune complexes in the tissue and removal of these complexes, mainly by neutrophils, were the predominant features of the RPAR observed by us. The RPAR was, however, not associated with any significant tissue damage or thrombosis. This, in fact, demonstrates that tissue can be cleared of immune complexes without the development of detrimental effects to the host.

We wish to thank Miss E. Januschke for her excellent technical assistance and Miss R. Bindeballe for performing the controls.

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Dr. K. Weber Dr. H. Ueki Dr. H. H. Wolff Prof. Dr. O. Braun-Falco Dermatologische Klinik und Poliklinik der Universit~t D-8000 Miinehen 2 Frauenlobstr. 9 Federal Republic of Germany

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Ultrastructural evidence for lack of tissue damage in a local immune complex reaction