JANICE B. ALLEN AND RONALD L. WILDER*. Arthritis and Rheumatism ..... We thank Dawn Smith for her assistance in preparing the manu- script and Edward ...
Vol. 55, No. 3
INFECTION AND IMMUNITY, Mar. 1987, p. 674-679
0019-9567/87/030674-06$02.00/0 Copyright X 1987, American Society for Microbiology
Variable Severity and Ia Antigen Expression in Streptococcal-CellWall-Induced Hepatic Granulomas in Rats JANICE B. ALLEN AND RONALD L. WILDER* Arthritis and Rheumatism Branch, National Institute of Arthritis, Musculoskeletal, and Skin Diseases, Bethesda, Maryland 20892 Received 25 September 1986/Accepted 25 November 1986 We have previously reported that a single intraperitoneal injection of an aqueous suspension of group A streptococcal cell wai (SCW) fragments induces extensive hepatic granulomas in LEW/N female rats, but not in F344/N female rats. To further understand the mechanisms underlying these differences, we compared granuloma development and class II major histocompatibility complex antigen (Ia) expression in histocompatible LEW/N, F344/N, and CAR/N female rats in response to SCW fragments of four different average molecular sizes. In LEW/N female rats, the smalest fragments (500 megadaltons) induced equivocal disease. Fragments of intermediate size induced granulomas of intermediate severity. The extent of granuloma development, the intensity of Ia antigen expression, and the amount of SCW antigen deposited in the liver qualitatively paralleled each other. In contrast, injection of the most granulomagenic SCW fragments into F344/N and CAR/N rats did not induce granulomas. Although these rat strains are histocompatible with the LEW/N (i.e., RTL.1) strain, hepatic Ia antigen expression in these strains was not increased significantly above basal levels. The amount of SCW antigen in the livers of the resistant rat strains appeared similar to the amount in the susceptible LEW/N strain. These data indicate that granuloma development is dependent on the size of the SCW fragment and host genetic background and that Ia expression directly parallels the severity of the hepatic disease. In addition, the data suggest that non-major histocompatibility complex genetic loci play a major role in regulating the development of the hepatic disease.
expression in the livers of LEW/N rats parallel the size of the granulomas and intensity of mononuclear cell infiltration. Moreover, we show that SCW fragments that are highly granulomagenic in the LEW/N female rat fail to induce granulomas or enhanced Ia antigen expression in the livers of F344/N and CAR/N females. These latter inbred strains have a major histocompatibility complex haplotype similar to that of the LEW/N rats.
A single systemic administration of an aqueous suspension of group A streptococcal cell wall (SCW) fragments into susceptible female rats results in the formation of granulomas in the liver (9, 19, 20). Within hours of injection, SCW fragments begin to deposit diffusely in the liver. Coincidentally the liver is infiltrated with polymorphonuclear and mononuclear leukocytes, as well as diffuse fibrin deposits. This acute reaction reaches maximum intensity at 3 to 5 days and then gradually subsides, but is followed by a chronic phase which becomes apparent approximately 3 weeks after SCW fragment injection and is associated with an influx of mononuclear cells, which form aggregates composed of T lymphocytes and macrophages (2, 4, 17, 18). The chronic phase develops as an apparent consequence of the persistence of the SCW fragment in the liver (2, 19). During the course of mononuclear cell organization, fibroblasts also infiltrate the area, apparently in response to T-lymphocyte and macrophage-derived factors. These cells tend to localize as a peripheral rim around the mononuclear cells, where they proliferate and generate type I and III collagen (17, 18). Over the next several months, the granulomas tend to shrink as they become less cellular and more fibrotic, but may persist indefinitely. In the present study, we have addressed the role of SCW fragment size on granuloma development and class II major histocompatibility complex antigen (Ia) expression in the liver. We demonstrate that granulomagenic activity in susceptible LEW/N rats is dependent on fragment size. We also demonstrate that the intensity and extent of Ia antigen *
MATERIALS AND METHODS Animals. Female specific-pathogen-free LEW/N, F344/N, and CAR/N rats, weighing approximately 100 g (approximately 6 weeks old) at the initiation of each experiment, were obtained from the Small Animal Section, National Institutes of Health, and maintained under specific-pathogen-free conditions as previously described in detail (2). Injection of SCW fragments. Group A SCW fragments were prepared as described previously (20). Briefly, SCW fragments were generated by sonication for 70 min at maximum frequency with a Biosonic IV sonicator fitted with a 3/4-in. (ca. 1.9-cm) probe (VWR Scientific, Baltimore, Md.). Fragments with mean molecular sizes of approximately 500 megadaltons (MDa) were isolated by centrifugation as described by Fox et al. (8). Sterile suspensions of SCW fragments in phosphate-buffered saline (pH 7.4) were injected intraperitoneally into the rats at a dose equivalent to 20 ,ug of SCW rhamnose per g of body weight (6). Sonicated but unfractionated SCW fragments at this dose induce hepatic granulomas in LEW/N rats with greater than 90% incidence (20). Control animals were injected with an equal volume of phosphate-buffered saline.
Corresponding author. 674
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STREPTOCOCCAL-CELL-WALL-INDUCED HEPATIC GRANULOMAS
Preparation of liver for histological evaluation. Specimens of liver were fixed in 10% buffered Formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Immunoperoxidase technique for SCW antigen detection. Formalin-fixed liver was embedded in paraffin, sectioned, and stained for group A SCW antigens by an immunoperoxidase technique (ABC Vectastain kit; Vector Laboratories, Burlingame, Calif.) as described previously (2, 10-11). A black precipitate identifies the SCW antigen. Controls included sections from saline-injected rats and SCW fragmentinjected rats stained with normal rabbit globulin. Specificity was verified by absorption of the antibody on group A SCW fragments, which completely eliminated positive staining. Immunoperoxidase staining technique for Ta antigen detection. Specimens of liver were snap frozen, sectioned, and stained for Ta antigens by an immunoperoxidase technique (ABC Vectastain Kit) as described previously (2, 10-11). Ia antigens were identified by using saturating amounts of a mouse monoclonal antibody against rat Ia antigen (OX6; Accurate Chemical Co., Westbury, N.Y.), which recognizes nonpolymorphic determinants on rat class TI major histocompatibility complex antigens. A black precipitate identifies the sites of Ia antigen expression. Control sections were stained with no primary antibody, an irrelevant monoclonal antibody, or mouse ascites fluid. The duration of all incubations was carefully controlled so that all tissues, which included appropriate diseased and control specimens on any particular day, were stained under identical conditions. Analysis of hepatic inflammatory response. All slides were coded for analysis. Hematoxylin-and-eosin-stained specimens were graded (0 to 4+) on the basis of granuloma size and extent. The location and intensity of cellular infiltration and fibrosis were also considered in grading the induced disease. Sections stained for SCW antigens were graded (0 to 4+) on the basis of extent and intensity of positive cytoplasmic staining. Sections stained for Ia antigens were graded (0 to 4+) in three anatomical sites: (i) in granulomata, (ii) in periportal regions, and (iii) in the parenchyma. Grading reflected both the frequency of positive cells (proportion of stained cells per total nuclei) and the relative overall intensity of positive staining. The livers were divided so that histological damage, antigen content, and Ia expression were assessed in the same rat. The data represent the mean of at least two separate experiments with three to five rats in which all livers were examined. RESULTS Histological observations. It is well documented that hepatic granulomas can be induced in young LEW/N female rats (6 weeks of age or less) by a single intraperitoneal injection of group A SCW fragments (4, 17-21), but the role of fragment size on lesion development has not been addressed. Thus, 6-week-old female LEW/N rats were given an intraperitoneal injection of SCW fragments, of four different approximate mean molecular sizes (500 MDa) at a dose of 20 ,ug/g of body weight. Since SCW-induced granulomas are usually well developed at 8 weeks, we sacrificed the animals at that time and examined their livers for evidence of any abnormalities. Substantial differences in histological appearance were observed (Fig. 1 and Table 1). The smallest fragments, 500 MDa, developed only equivocal evidence of an inflammatory response, characterized primarily by increased periportal lymphocytic cellularity (Fig. 1D). The inflammatory response elicited by fragments of intermediate size (-50 and -500 MDa) are shown in Fig. 1B and 1C. Small aggregates of inflammatory mononuclear cells were elicited throughout the liver in rats that received the 50-MDa fragments (Fig. 1B) and still fewer and smaller granulomas in rats that received the 500-MDa fragments (Fig. 1C). SCW antigen distribution. To address the possibility that the larger-sized fragments did not localize in the liver as an explanation for the lack of granuloma-inducing activity, we analyzed tissue sections for the presence of SCW antigens by immunoperoxidase staining techniques. SCW antigens were detected in all of the SCW-injecting LEW/N rats, but the severity of granuloma development paralleled the amount of antigen in the tissue, as assessed qualitatively by staining intensity (Fig. 2 and Table 1). Figure 2A shows a section of liver from a representative LEW/N rat injected with the smallest fragments (500 MDa) clearly had less SCW antigen in their liver (Fig. 2D). Livers from uninjected LEW/N rats showed no positive staining (data not shown). We conclude that the absence of an inflammatory response was not simply due to failure of the SCW antigens to distribute into the liver, but may correlate with the amount of antigen deposited. Ia antigen expression in the liver. Since Ia antigen expression is believed to be a central event in initiating and sustaining a cell-mediated immunopathologic response (15-16), we studied the expression of Ta in the livers of LEW/N rats injected with SCW fragments of the four different sizes and compared it with basal levels of Ia antigen expression. The intensity of Ta antigen expression was inversely correlated with the size of SCW fragment injected (Fig. 3 and Table 1). Ta antigen was intensely expressed in the granulomas and periportal regions and throughout the parenchyma of rats injected with the smallest fragments (500 MDa) and shows almost background levels of Ia staining. These findings indicate that enhanced hepatic Ta antigen expression parallels disease severity and is inversely dependent on the size of the SCW fragments. Rat strain dependence. Since we have previously reported that F344/N female rats do not develop hepatic granulomas after injection with SCW fragments, we reexamined this rat strain for granuloma development after injecting the most granulomagenic SCW fragments (500 MDa, induced a minimal inflammatory response. The fragments of intermediate size, approximately 50 or 500 MDa, induced small and less extensive granulomas. Moreover, we have demonstrated an apparent inverse relationship between the relative amount and distribution of SCW antigen deposited in the liver and fragment size that parallels the severity of the induced inflammatory reaction. Our results are reminiscent of those reported by Fox et al. with regard to the arthritis that also develops after SCW 1.
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FIG. 3. Demonstration of Ia antigens in LEWIN female rats 8 weeks after intraperitoneal injection of SCW fragments (magnification, x 62). The black precipitate identifies the Ia antigens. Sections from representative LEW/N rats receiving the smallest fragments (500 MDa) (C).
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FIG. 4. Demonstration of group A SCW antigens in (A) F344/N and (B) CAR/N female rats 8 weeks after intraperitoneal injection of the smallest (
