Toward understanding the role of cartilage particulates in synovial inflammation.

OBJECTIVE: Arthroscopy with lavage and synovectomy can remove tissue debris from the joint space and the synovial lining to provide pain relief to patients with osteoarthritis (OA). Here, we developed an in vitro model to study the interaction of cartilage wear particles with fibroblast-like synoviocytes (FLS) to better understand the interplay of cartilage particulates with cytokines on cells of the synovium. METHOD: In this study sub-10 µm cartilage particles or 1 µm latex particles were co-cultured with FLS ±10 ng/mL interleukin-1α (IL-1α) or tumor necrosis factor-α (TNF-α). Samples were analyzed for DNA, glycosaminoglycan (GAG), and collagen, and media samples were analyzed for media GAG, nitric oxide (NO) and prostaglandin-E2 (PGE2). The nature of the physical interaction between the particles and FLS was determined by microscopy. RESULTS: Both latex and cartilage particles could be phagocytosed by FLS. Cartilage particles were internalized and attached to the surface of both dense monolayers and individual cells. Co-culture of FLS with cartilage particulates resulted in a significant increase in cell sheet DNA and collagen content as well as NO and PGE2 synthesis compared to control and latex treated groups. CONCLUSION: The proliferative response of FLS to cartilage wear particles resulted in an overall increase in extracellular matrix (ECM) content, analogous to the thickening of the synovial lining observed in OA patients. Understanding how cartilage particles interface with the synovium may provide insight into how this interaction contributes to OA progression and may guide the role of lavage and synovectomy for degenerative disease.

The ontogeny of antigen-specific T cells.

Although the fetal lamb is unable to form circulating antiovalbumin antibodies until about 120 days of gestation, ovalbumin-specific helper T cells can be stimulated to function at an earlier age. This would suggest that the critical event responsible for the precise sequential maturation of immunologic competence to different antigens at different developmental stages is not the first appearance of specific receptors on immunocytes. Alternative explanations of this phenomenon are discussed.

Nonspecific signals for B-cell localization and activation.

Virgin, inactive mammary gland autografted to the anterior chamber of the rabbit eye remains free of lymphoid cells. Activation of the ectopic gland by systemic injection of chorionic gonadotropin results in maturation of the gland and milk production, accompanied by the immirgration of lymphocytes and their activati-n to Ig formation, predominantly of the IgA class. In the presence of antigen-induced intraocular inflammation, the activated gland is able to influence the Ig class of B cells in the neighboring ocular tissues. These data suggest that even nonlymphoid tissues may elaborate lymphocyte-homing and polyclonal B-cell activating factors which function independently of specific antigen.

Visna virus infection of American lambs.

Random-bred fetal and 4-week-old American lambs, inoculated intracerebrally with visna virus, developed a persistent infection in the brain and sometimes in the lung. The pathologic changes present in these lambs were similar to the early lesions of visna in Icelandic sheep, thus providing a possible model for the study of virus-induced demyelinating disease.

A model for the cytoplasmic trafficking of signalling proteins involving the hsp90-binding immunophilins and p50cdc37.

A number of transcription factors and protein kinases involved in signal transduction exist in heterocomplexes with the ubiquitous and essential protein chaperone hsp90. These signalling protein x hsp90 heterocomplexes are assembled by a multiprotein chaperone system comprising hsp90, hsp70, Hop, hsp40, and p23. In the case of transcription factors, the heterocomplexes with hsp90 also contain a high molecular weight immunophilin with tetratricopeptide repeat (TPR) motifs, such as FKBP52 or CyP-40. In the case of the protein kinases, the heterocomplexes contain p50cdc37. The immunophilins bind to a single TPR acceptor site on hsp90, and p50cdc37 binds to an adjacent site so that binding is exclusive for p50cdc37 or an immunophilin. Direct interaction of immunophilins with the transcription factors or p50cdc37 with the protein kinases leads to selection of different heterocomplexes after their assembly by a common mechanism. Studies with the glucocorticoid receptor, for which translocation from the cytoplasm to the nucleus is under hormonal control, suggest that dynamic assembly of the heterocomplexes is required for rapid movement of the receptor through the cytoplasm along cytoskeletal tracts. As for the similar short-range trafficking of vesicles along microtubules, there must be a mechanism for linking the signalling protein solutes to the molecular motors involved in movement. We present here a model in which the immunophilins and p50cdc37 target, respectively, the retrograde or anterograde direction of signalling protein movement by functioning as connectors that link the signalling proteins to the movement machinery.

Sindbis virus-induced ocular immunopathology.

The intraocular injection of the Sindbis virus in adult BALB/c mice produces a uveoretinitis with little or no central nervous system involvement. Ocular disease starts on the third day after infection and presents as a mild to moderate iridocyclitis and retinitis, usually accompanied by typical severe dysplastic changes of the retina. The inflammatory infiltrate consists almost exclusively of lymphocytes and histiocytes. Immunosuppression of the mouse with cyclophosphamide on the day after infection markedly reduces or eliminates completely the inflammatory response, suggesting that the virus itself is not cytopathogenic. In the normal host, the virus replicates within the eye for several days but is then completely eliminated by day 8 after infection. In the immunosuppressed animal, virus titers reach greater levels than in the normal animal and then fall, in step with the developing inflammatory response. It would appear that the immunologic mechanisms responsible for clearance of the viral infection from the eye also mediate the ocular disease.

Mechanisms of allograft rejection of corneal endothelium.

The local intraocular graft-vs.-host (GVH) reaction, involving the destruction of the corneal endothelial cells of the rabbit host by sensitized donor lymphoid cells, has been used to study the mechanism of corneal allograft rejection. Pretreatment of donor cells with a specific mouse monoclonal hybridoma anti-T cell antibody and complement suppresses the destructive reaction, suggesting that a cellular-immune mechanism is primarily involved. Pretreatment of donor cells with mitomycin-C completely abolishes the local GVH reaction, indicating that the effector lymphocytes must undergo mitosis within the eye before they can engage in target cell destruction. Finally, studies of the local GVH reaction in irradiated leukopenic recipients or in preinflamed rabbit eyes suggest that host leukocytes may contribute nonspecifically to enhance the destructive process. These studies show that the local ocular GVH reaction may provide a useful model for the study of the mechanisms involved in the rejection of corneal allografts.

Experimental autoimmune dacryoadenitis. I. Lacrimal gland disease in the rat.

Experimental autoimmune dacryoadenitis was produced in Lewis rats by immunization with a single intradermal administration of a 3M KCl extract of exorbital lacrimal gland in CFA, when enhanced by simultaneous i.v. injection of killed Bordetella pertussis. No significant lacrimal lesions were observed in control animals immunized with the extracts of Harderian or salivary glands. Gel filtration of the 3M KCl extract on Sephacryl S-300 column yielded three protein fractions. Only fraction III (MW = 10-55K) induced marked dacryoadenitis following a single injection of 2.0 mg protein in CFA plus pertussis. The infiltrates in the exorbital lacrimal lesions were first apparent around the ducts and associated vasculature. From this area, the infiltrates appeared to spread to the acini drained by these ducts, ultimately involving as much as 30-50% of the gland. The affected glands most commonly showed a diffuse nongranulomatous infiltrate of small lymphocytes, macrophages, and plasma cells; this was focal in nature, involving acinar atrophy and breakdown, and replaced the normal architecture in extreme cases. The Harderian and salivary glands were uninvolved in these animals, suggesting a restricted specificity of this response. Lewis rats immunized with exorbital lacrimal gland fractions I or II in CFA plus pertussis showed only minimal lesions, similar to controls receiving CFA and pertussis without antigen. These findings suggest that an autoantigen exists in the lacrimal gland of the rat that is capable of inducing a specific lymphoproliferative dacryoadenitis.

Experimental autoimmune dacryoadenitis. II. Harderian gland disease in the rat.

Experimental autoimmune dacryoadenitis was induced in 100% of Lewis rats by immunization with a KCl extract of Harderian gland in complete Freund's adjuvant (CFA), providing that the animals had received simultaneously i.v. injection of killed Bordetella pertussis. No significant pathological changes in the Harderian gland were observed in control animals immunized with KCl extracts of lacrimal or salivary glands. Gel filtration of the KCl extract on Sephacryl S-300 column yielded three protein fractions. Fraction II (MW = 50-100K) induced severe Harderian gland disease following a single injection of 2.0 mg protein in CFA plus pertussis. The initial lesions consisted of multiple focal infiltrates of mononuclear cells. Later, the inflammatory process assumed a more granulomatous form, with significant contribution by epithelioid and giant cells. In contrast, Lewis rats immunized with Harderian gland fractions I or III proteins, or with extracts of lacrimal or salivary gland, showed little or no inflammatory lesions. These data suggest that Harderian gland contains unique tissue-specific autoantigen(s) capable of inducing autoimmune granulomatous dacryoadenitis in the rat.

Conjunctival immunity: compared effects of ocular or intestinal immunization in rats.

The ability to induce a conjunctival antitoxin response by conjunctival or enteric administration of cholera toxin antigen was studied in rats. Repeated enteric immunization caused a vigorous jejunal antitoxin response, but none in the conjunctiva. Enteric immunization did, however, prime for a conjunctival antitoxin response to locally applied antigen, as did direct ocular administration of cholera toxin. Vigorous conjunctival antitoxin responses occurred only after ocular challenge, and were localized to the challenged eye. These results agree with the notions that (1) specific memory cells migrate to the conjunctiva after enteric immunization, or arise locally after ocular immunization; and (2) specific antibody-producing plasma cells arise almost entirely within the immunized conjunctiva, and few if any migrate to the conjunctiva from distant mucosae or from the conjunctiva of the immunized eye to that of the nonimmunized eye.

In vitro enhancement of anterior chamber GVH reactions.

The injection of sensitized allogeneic lymphocytes into the anterior chamber of the rabbit eye results in a local graft-verus-host reaction, with focal destruction of the corneal endothelium. This experimental model permits in vitro manipulation of effector cells, and the study of the mechanisms involved in corneal graft rejection. The authors now show that the in vitro activation of sensitized lymphocytes is a one-way mixed lymphocyte reaction yields "supersensitized" effector cells which are quantitatively enriched and qualitatively altered to yield more severe and more rapid endothelial target cell destruction.

Secretory immune system of rabbit ocular adnexa.

A distinct system of immunity in a variety of animals is located subjacent to epithelial surfaces and is typified by the predominance of immunoglobulin A (IgA) and secretory component (SC) in various external secretions, including tears. The present study examined normal rabbit lacrimal gland, conjunctiva, and cornea for the presence of immunoglobulin and SC. IgA-staining plasma cells predominated within lacrimal gland and conjunctival stroma, and SC was found in the epithelial cells of both these tissues but not within corneal epithelium. These observations are consistent with findings for other secretory sites in both rabbits and humans and establish lacrimal gland and conjunctiva as integral parts of the rabbit secretory immune system.

Experimental immunogenic granuloma of the orbit: transfer of granulomatous hypersensitivity with a subset of T lymphocytes.

An experimental model to investigate orbital granuloma formation in inbred rats was established. Animals sensitized to trinitrophenyl ovalbumin (TNP-OA) and challenged retro-orbitally with TNP-OA covalently linked o Sepharose 4B beads specifically developed a granulomatous response. This granulomatous reactivity was passively transferred into normal animals by lymph node cells, but not by serum antibody from sensitized donors. Lymphocytes which transfer granuloma formation in normal recipients were characterized by cell fractionation and membrane marker analysis. These experiments show that the effector cells capable of transferring granulomatous hypersensitivity are enriched in the lower density fractions on discontinuous Percoll gradients. These cells are lymphoblasts and express the W3/25 helper T lymphocyte marker. It was also demonstrated that lymphoid cells from sensitized donors in the higher density Percoll fraction appear to be incapable of adoptively transferring granulomatous responsiveness directly to normal recipients. However, incubation of these high density lymphocytes with specific antigen resulted in marked enhancement of their ability to transfer the disease. Antigen-induced activation also resulted in an increase in both lymphoblasts and the W3/25 marker. The authors conclude, therefore, that a subset of T cells which are lymphoblasts and express the helper-cell marker is responsible for granuloma formation in sensitized animals and is capable of transferring orbital granuloma formation to non-sensitized normal recipients.

The role of lymphokines in immunogenic uveitis.

Lymphokines were prepared from rabbit lymph node cells specifically activated in vitro with the insoluble antigen ovalbumin or nonspecifically activated with the insoluble mitogen concanavalin A. Intravitreal injection of either lymphokine caused uveitis in the normal rabbit eye, more severe and more prolonged with the concanavalin A-activated supernatants than with the ovalbumin-activated supernatants. The most striking difference between the two lymphokine preparations was in their ability to induce a secondary intraocular antibody response in the trinitrophenyl bovine gamma globulin-primed recipient. Concanavalin A supernatants stimulated polyclonal B cell activation with appreciable anti-TNP responses in animals primed either systemically or locally. In contrast, ovalbumin supernatants stimulated an intraocular anti-TNP response only in those animals that had been primed within the eye. We speculate that two or more different lymphokines are active, some of which attract lymphocytes nonspecifically to the local site and others of which excite polyclonal B cell activation and plasma cell formation.

Desquamation of corneal endothelial cells.

The process of wound healing of rabbit corneal endothelium involves the desquamation of significant numbers of endothelial cells, which may be found floating freely in the aqueous. It is suggested that this may be a general phenomenon of corneal endothelial wound healing, which accompanies the release of endothelial-cell attachments to Descemet's membrane and to neighboring cells during mitosis, allowing some of the cells to "fall of" into the anterior chamber. The possibility is discussed that such endothelial desquamation may contribute to had sensitization to he histocompatibility antigens of penetrating corneal grafts.

Secretory component of IgA: a marker for differentiation of ocular epithelium.

Secretory component (SC) was studied by indirect immunofluorescence of the ocular surface epithelium. We find that conjunctival epithelium produces this component, and that it is absent in the corneal epithelium. Conjunctival epithelium loses its SC staining within 1 to 2 days as it grows over a denuded corneal stroma. This implies a very rapid turn-off of the SC gene and rapid export of the gene product previously formed. However, conjunctival flap epithelium does not change its characteristic structure or functions. Vascularization of corneas resurfaced by conjunctival epithelium usually leads to a rapid reversal of both morphological and biochemical characteristics in the surface epithelium, as judged by the reappearance of goblet cells and positive staining for SC. Vascularization of normal corneas leaves the epithelium unchanged, so that neither goblet cells nor SC appear. Thus metaplasia of conjunctival to corneal epithelium is incomplete, permitting reversion to type under certain conditions.

An animal model for cicatrizing trachoma.

An animal model of cicatrizing trachoma was developed in cynomolgus monkeys. This model is consistent with our hypothesis that repeated ocular inoculation of Chlamydia trachomatis, BOUR strain, mimics the repeated reinfection that occurs naturally in endemic human trachoma. A chronic follicular conjunctivitis developed, and scarring later appeared in the superior tarsal conjunctiva. The organism was reisolated after the infection and was also demonstrated cytologically. Specific antichlamydial antibodies of both the IgM and IgG types appeared in the sera of the monkeys. Histopathologic examination of conjunctiva showed a marked lymphocytic response and the presence of germinal centers; areas of conjunctival scar tissue were also examined. Efforts to produce a similar model in rhesus monkeys were less successful.

An animal model of trachoma II. The importance of repeated reinfection.

An animal model of chronic cicatrizing trachoma has been produced by repeated ocular inoculation with Chlamydia trachomatis serotype E, a genitally transmitted strain. We have now produced a chronic follicular conjunctivitis on cynomolgus monkeys by repeated inoculation with C. trachomatis serotype A, which has been isolated from an area of endemic trachoma. This disease was similar in all respects to that which followed infection with the serotype E strain. Cynomolgus monkeys inoculated with a single dose of serotype E of C. trachomatis strain developed an acute, self-limited follicular conjunctivitis, which was intense for 4 weeks and then slowly subsided. The organism could be reisolated only during the first 4 weeks after inoculation. On reinoculation at 15 and 30 weeks after the initial infections, these animals demonstrated only a mild and transitory clinical response, and the agent could be recovered for only up to 14 days after inoculation. In contrast, repeated weekly reinoculation with either serotype led to a chronic progressive clinical response in these animals, although after the first 6 weeks the agent was isolated only occasionally. This chronic disease was shown not to be due to hypersensitivity to the egg yolk components in which the organism was grown. These data suggest that the serotype of the chlamydial organism may not be as important in determining the clinical course of disease as is the frequency or persistence of exposure to the chlamydial agent. Although a single inoculum produced an acute follicular conjunctivitis, repeated inoculation is needed to produce the chronic disease characteristic of trachoma in this animal model.