Systemic anti-tumor necrosis factor antibody treatment exacerbates endotoxin-induced uveitis in the rat.

Tumor necrosis factor is released in the circulation and aqueous humor during endotoxin-induced uveitis, and induces acute uveitis when injected intraocularly in rats. To elucidate the role of tumor necrosis factor in the development of endotoxin-induced uveitis we analysed the effect of neutralizing anti-tumor necrosis factor antibodies and of pentoxifylline, a drug that inhibits tumor necrosis factor synthesis. Lewis rats were treated with: (a) a single intracardial injection of polyclonal rabbit anti-murine tumor necrosis factor antiserum prior to foot pad injection of 200 micrograms lipopolysaccharide; (b) an intraperitoneal injection of 10 mg pentoxifylline 1 hr before, at the time of, and 3 hr after foot pad injection of lipopolysaccharide; or (c) an intravitreal injection of 20 to 500 micrograms pentoxifylline together with 1 microgram lipopolysaccharide. The ocular inflammation was examined by slit-lamp and evaluated for the presence of hyperemia, flare, miosis, infiltrating cells or hypopyon. Levels of tumor necrosis factor in serum and aqueous samples were determined using a bioassay. Systemic treatment with either anti-tumor necrosis factor antibodies or pentoxifylline resulted in a significant inhibition, 90 and 70% respectively, of serum tumor necrosis factor activity at 3 to 4 hr after lipopolysaccharide injection. Systemic pentoxifylline treatment had no influence on the severity of uveitis. Anti-tumor necrosis factor antibody treatment, in contrast, caused an exacerbation of endotoxin-induced uveitis at t = 20 hr; mean uveitis score 3.9 vs. 1.4 in controls; P < 0.01. Intraocular administration of pentoxifylline together with lipopolysaccharide also had an aggravating effect on uveitis, that was associated with increased levels of intraocular tumor necrosis factor. The results show that inhibition of serum tumor necrosis factor activity does not block the development of endotoxin-induced uveitis. In fact, anti-tumor necrosis factor antibody treatment exacerbates the intraocular inflammation. These findings suggest that tumor necrosis factor may have other than proinflammatory properties in this uveitis model.

Tumour necrosis factor-induced uveitis in the Lewis rat is associated with intraocular interleukin 6 production.

Lewis rats were injected with recombinant murine tumour necrosis factor-alpha either intravitreally (0.08-50 ng) or intracardially (1 microgram). The intraocular inflammatory response induced by tumour necrosis factor was examined by slit-lamp and protein extravasation into aqueous humor was determined. The phenotype of the inflammatory cells in the eye was analysed by immunohistochemistry. In addition, the kinetics of intraocular interleukin 6 production were determined. At 24 hr after intravitreal injection, a significant clinical uveitis was observed only in rats injected with 50 ng of tumour necrosis factor, when compared to saline-treated controls (P < 0.05). Maximal clinical uveitis and blood-aqueous barrier breakdown were already present at 4 hr after tumour necrosis factor injection. The uveitis was characterized by a massive cellular infiltrate in the anterior segment, consisting predominantly of polymorphonuclear cells and macrophages/monocytes, and to a lesser extent of T lymphocytes. Intraocular interleukin 6 mRNA expression and elevated levels of interleukin 6 in aqueous humor were detected 1 hr after tumor necrosis factor injection, reached a maximum at 3 to 4 hr after injection, and had declined again at 2 hr. Although intracardial injection of 1 microgram of tumour necrosis factor in Lewis rats induced a rise of circulating interleukin 6, it did not produce uveitis. The results obtained with intravitreally injected tumour necrosis factor indicate that intraocular TNF may play a pivotal role in the induction of uveitis in the rat. The transient intraocular production of interleukin 6 early during tumour necrosis factor-induced uveitis suggests that this cytokine may participate in the response induced by tumour necrosis factor.

Kinetics of intraocular tumor necrosis factor and interleukin-6 in endotoxin-induced uveitis in the rat.

PURPOSE: To determine the kinetics of tumor necrosis factor (TNF) and interleukin-6 (IL-6) in serum and aqueous humor of rats with different susceptibilities to endotoxin-induced uveitis (EIU), after footpad injection of lipopolysaccharide (LPS). METHODS: Samples were collected from EIU-susceptible Lewis rats and EIU-resistant Brown Norway (BN) rats for up to 72 hours after LPS injection. Specific bioassays were used to measure TNF and IL-6 activity. Northern blot analysis was used to assess intraocular IL-6 mRNA expression. RESULTS: High levels of TNF and IL-6 were detected in serum of both rat strains early after LPS injection. A second rise in serum TNF was observed at 18 to 20 hours in Lewis rats only. In aqueous humor of Lewis rats, high levels of TNF and IL-6 were observed early after LPS injection (2 to 8 hours) and concomitant with maximal uveitis (18 to 24 hours). Low levels of TNF and IL-6 were found in aqueous humor of BN rats. Ocular IL-6 mRNA was detected at the same time as IL-6 activity was measured in aqueous humor. CONCLUSIONS: The results of this study indicate that both TNF and IL-6 may play a role in the pathogenesis of EIU. The early release of TNF in aqueous humor during EIU suggests that this cytokine may serve as an initial mediator of intraocular inflammation. Furthermore, Northern blot analysis indicates that IL-6 is produced locally during EIU.

Analysis of IL-6 levels in human vitreous fluid obtained from uveitis patients, patients with proliferative intraocular disorders and eye bank eyes.

Several studies suggest a role for IL-6 in the pathogenesis of uveitis. Earlier we have shown that aqueous humour obtained from patients with uveitis contained raised levels of IL-6. In the study described here we investigated the IL-6 levels in vitreous fluid samples obtained from 75 uveitis patients with different uveitis entities. Vitreous samples from 14 patients with proliferative intraocular disorders (PID) and 29 eye bank eyes were used as controls. All the samples were tested in the IL-6 B9 bioassay as well as in a sensitive ELISA for IL-6. Raised IL-6 levels were detected in the vitreous fluid of uveitis patients as well as patients with PID, implicating IL-6 as a common inflammatory mediator. The highest mean level of IL-6 was found in the vitreous fluid of patients with acute retinal necrosis. The mean IL-6 levels measured by the ELISA were higher compared to the levels measured by the B9 bioassay. This may be caused by the presence of B9 bioassay inhibitory factors in the vitreous fluid of these patients.

Analysis of aqueous humour in ocular onchocerciasis.

The development of an onchocercal chorioretinopathy from the first detectable signs to a full blown oncho fundus is not fully understood. We investigated the intraocular humoral immune response against Onchocerca volvulus, human S-antigen, IRBP and crude retinal extract (using an ELISA) by examining paired aqueous humour and serum samples obtained from onchocerciasis patients (without [n = 10] and with ocular symptoms [n = 8]) and endemic controls [n = 14] from Sierra Leone (West Africa). A local intraocular anti-retinal IgG antibody production could not be demonstrated in onchocerciasis patients, whether they had ocular symptoms or not. A significantly higher level of O. volvulus antibodies and IgG was measured in the aqueous of onchocerciasis patients with ocular involvement, as compared to patients without ocular symptoms (Mann-Whitney ranksum test; p less than 0.001 and p less than 0.02 respectively). Since interleukin-6 (IL-6) plays an essential role in the differentiation of B cells into immunoglobulin producing plasma cells, we therefore measured this cytokine in paired aqueous and serum samples. Elevated IL-6 levels were found in the aqueous of two out of eight onchocerciasis patients tested. In view of these findings it seems improbable that retinal autoimmunity is a major factor in the pathogenesis of onchocercal chorioretinopathy. The high intraocular levels of antibodies against the parasite suggest a direct involvement of the parasite in the pathogenesis of onchocercal chorioretinopathy.

Analysis of interleukin-6 in endotoxin-induced uveitis.

The mechanisms underlying the induction of intraocular inflammation in the rat model of endotoxin-induced uveitis (EIU) and the subsequent development of tolerance after repeated endotoxin injections are poorly understood. Interleukin-6 (IL-6) was measured in the aqueous humor and serum of Lewis rats after single and repeated injections of endotoxin into the footpad. After a single injection, a rise in serum and aqueous-humor levels of IL-6 was seen after 2 and 16 hr, respectively. The highest aqueous-humor level of IL-6 was seen 20 hr postinjection and was tenfold that seen in the serum sample taken at the same time, suggesting intraocular synthesis of this cytokine. Four hours later the most active uveitis and the highest total aqueous-humor protein level were observed. Repeated injection of endotoxin still resulted in a moderate but significant systemic release of IL-6 but no detectable IL-6 in the aqueous humor and the absence of uveitis. Intravitreal injection of endotoxin-free human recombinant IL-6 (10-10(5) U) in rats resulted in uveitis, resembling the ocular response to endotoxin. There appeared to be a prozone effect regarding the total aqueous-humor protein concentration. The largest amount of aqueous-humor protein was seen in the eyes injected with 10(2) U of IL-6, but increasing concentrations of intravitreal IL-6 showed a corresponding decrease in protein levels. In the fellow saline-injected eyes, a clear consensual response was observed with regard to the extravasation of protein, although the uveitic grade in these eyes was low or zero. Repeated intravitreal injection of IL-6 resulted in ocular unresponsiveness in nine of 11 rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Indomethacin increases the sensitivity of the monocyte migration inhibition assay.

In ophthalmo-immunological investigations only small samples of ocular tissues and fluid are available and assays which are feasible with very small volumes or cell numbers are mandatory. Indomethacin, which is known to augment the immune response both in vivo and in vitro was therefore tested for its effect on the monocyte migration inhibition (MIF) assay using low cell or antigen doses. The sensitivity of the MIF assay may be greatly increased by adding indomethaci during the first step of the assay. Titration of either the antigen dose, the mononuclear cells number or both per assay, resulted in a 10-50-fold increase in sensitivity of the assay, with a broad inter-individual variability. Increasing the sensitivity of the MIF assay with indomethacin has clear advantages with regard to the number of cells required but also confronts us with a new problem: activation of specific cells that circulate at very low frequencies in non-immunized individuals. The enhanced response could be reversed to some extent by adding prostaglandin E2 together with indomethacin to the first step of the assay. Moreover, adding leukotriene B4 to the first step of the assay had an enhancing effect over a limited concentration range. We conclude that in the presence of indomethacin, the MIF assay provides a highly sensitive technique for the demonstration of cellular immune responses in small samples of biological fluids containing very small numbers of antigen-specific lymphocytes.

Serum antibody response to human and bovine IRBP in uveitis.

Interphotoreceptor retinoid binding protein (IRBP) is a 136,000 molecular weight photoreceptor cell protein capable of inducing an experimental autoimmune uveitis (EAU) in susceptible animal strains. The occurrence of serum antibodies against human (Hu) or bovine (Bo) IRBP was investigated in patients with uveitis and healthy controls. A sensitive ELISA detected anti-IRBP in approximately 50% of patients and controls, without apparent differences in the mean level, titre or avidity and irrespective of the origin of the antigen. Although the correlation (p less than 0.001) between anti-HuIRBP and anti-BoIRBP levels in uveitis sera suggested the presence of crossreacting antibodies, these sera also contained antibodies specific for either the human or the bovine antigen. The only difference between patients and controls was the greater ability of antibodies in uveitis sera (p less than 0.05) to recognize a synthetic peptide of HuIRBP, which induces severe EAU in rats. We conclude that autoantibodies to IRBP occur naturally in man and are not increased in patients with uveitis.

Aqueous humor interleukin-6 levels in uveitis.

The level of Interleukin-6 (IL-6) in the aqueous humor of 24 patients with 2 types of uveitis was measured with a specific bioassay using the murine hybridoma cell line B9. Sixteen patients had Fuchs' heterochromic cyclitis (FHC) and 8 had toxoplasma uveitis (TU). Sixty-three percent of each of the FHC and TU groups had raised levels of IL-6 in their aqueous (mean: 543 and 19,228 units/ml respectively). Thirteen control aqueous samples, obtained at surgery for senile cataract, showed IL-6 levels of less than 10 units/ml. Serum obtained at the same time as each aqueous humor sample also showed IL-6 levels of less than 10 units/ml, indicating that the raised levels of IL-6 found in the aqueous of uveitis patients did not result from serum leakage, but from local production. This is the first report on intraocular IL-6 levels, and indicates that IL-6 may play a role as an inflammatory mediator in uveitis.

Aqueous humour analysis in Fuchs' heterochromic cyclitis.

Aqueous humor from 23 patients with Fuchs' heterochromic cyclitis (FHC) was analysed by a number of immunological methods. Intraocular IgG synthesis was found in 65% of patients and oligoclonal IgG bands, mainly of the IgG1 subclass, identified in 57%. There was a relative increase in IgG1 (P less than 0.01) as compared to patients with senile cataract. Local production of the cytokine Interleukin-6 was demonstrated in 63% of patients (P less than 0.01). Analysis of aqueous by HPLC and SDS-PAGE failed to reveal any abnormalities specific for FHC. These findings add further evidence to the theory of immune dysregulation in this condition.