D C Dumonde

Cytokines in proliferative vitreoretinopathy

This study determined the presence of interleukin 1 (IL-1), interleukin 6 (IL-6), tumour necrosis factor α (TNFa), tumour necrosis factor β (TNFβ), interferon γ (IFNγ), transforming growth factor β2 (TGFβ2) and fibroblast proliferation activity (FPA) in vitreous aspirates from eyes undergoing vitrectomy for the treatment of retinal detachment complicated by proliferative vitreoretinopathy (PVR) or uncomplicated retinal detachment (RD). Cadaveric vitreous from normal subjects were used as controls. The results showed that IL-1 and IL-6 predominated in vitreous from eyes with PVR or RD, and that concentrations of IL-6 >20 pg/ml were more frequently found in PVR than in RD (p = 0.031) or control specimens (p = 0.006). Low levels of TNFa were observed in 4/18 eyes with PVR, 1/15 eyes with RD and 1/15 control vitreous, and small concentrations of TNFα were seen in 3/18 eyes with PVR, 1/15 eyes with RD and 2/15 control vitreous. IFNγ was detected in 12/18 eyes with PVR, but only in 5/15 eyes with RD (p = 0.048) and 6/15 control specimens. TGFβ2 was present in all vitreous samples at concentrations ranging from 100 to 4,500 pg/ml with no significant differences among the three groups. Control vitreous possessed the greatest FPA when compared with vitreous from eyes with PVR (p = 0.031) or RD (p = 0.048). These observations provide further evidence that cytokine-mediated pathways of inflammation are involved in the pathogenesis of PVR and point to the possible involvement of IL-1, IL-6 and IFNγ in cellular interactions leading to chronicity.

Distribution of TNF alpha and its reactive vascular adhesion molecules in fibrovascular membranes of proliferative diabetic retinopathy.

AIMS: This study investigated the presence of the cytokine tumour necrosis factor alpha (TNF alpha) and the vascular adhesion glycoproteins ICAM-1, VCAM-1, E-selectin, P-selectin, and PECAM within fibrovascular membranes of eyes with proliferative diabetic retinopathy (PDR). METHODS: The presence of these molecules was determined by immunohistochemical staining using monoclonal antibodies and the APAAP technique. RESULTS: Staining for TNF alpha was observed on the retinal vascular endothelium of five of 12 specimens, on infiltrating cells within all membranes, and on the extracellular matrix of nine specimens. This staining wa abolished by absorption of the monoclonal antibody with human recombinant TNF alpha. Likewise, ICAM-1 staining was given by infiltrating cells and extracellular matrix of nine membranes and by the endothelium of three of the specimens. VCAM-1, E-selectin, and P-selectin staining was observed on the vascular endothelium of 5/12, 4/12, and 3/12 epiretinal membranes respectively. PECAM was expressed by the endothelium of 4/12 specimens, by infiltrating cells of 8/12 membranes, and also by the extracellular matrix of two of the specimens. CONCLUSION: The widespread distribution of TNF alpha and the nature of the adhesion molecules expressed by vascular endothelial cells in PDR membranes suggest that local activation of TNF alpha and enhanced expression of vascular cell adhesion molecules may play an important role in the development of the proliferative phase of diabetic retinopathy.

Heat shock protein peptides reactive in patients with Behçet's disease are uveitogenic in Lewis rats

Mycobacterial and homologous human heat shock protein T cell peptide epitopes specific for T lymphocytes in Behçets disease were investigated for their pathogenicity in Lewis rats. The potential pathogenicity of eight peptides and two controls was assessed by administering the peptides in enriched Freunds adjuvant into the footpads of male Lewis rats. Anterior uveitis which is a major manifestation of Behçets disease was induced with two out of the four mycobacterial and all four homologous human peptides. The most effective peptides inducing indocyclitis in 64‐75% of rats were peptides with amino acids 336‐351 and 136‐150, derived from the sequence of the human 60‐kD heat shock protein. A few of the rats also showed evidence of focal loss of photoreceptors. These results suggest that selected peptides within heat shock protein 60 kD which function as T cell epitopes in Behçets disease are capable of inducing uveitis in rats. This supports the view that the peptide T cell determinants may be involved in the pathogenesis of Behçets disease.

Distribution of cytokine proteins within epiretinal membranes in proliferative vitreoretinopathy.

This study reports on the immunohistochemical staining for cytokine proteins of 26 epiretinal membranes obtained from eyes undergoing surgery for the treatment of proliferative vitreoretinopathy. All specimens were investigated for the distribution of staining for interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) and interleukin-2 (IL-2). The results showed that 22 of the membranes (85%) stained for TNF alpha not only intracellularly but also in the extracellular matrix. This contrasts with the findings that only 2 membranes stained for IL-1 alpha and that another 3 were positive for IL-1 beta. Staining for the cytokines IL-6 and IFN gamma was also observed in 9 and 7 membranes respectively. None of the specimens investigated stained with antibodies to IL-2 or control antibodies, and none of three normal retinas stained with any of the antibodies used. Pre-absorption of anti-cytokine antibodies with the corresponding human recombinant cytokines abolished staining of cells and extracellular matrix. The present findings support growing evidence that cytokine-mediated pathways of inflammation are involved in the pathogenesis of proliferative vitreoretinopathy, and draw attention to the possibility that interaction between extracellular matrix-bound cytokine and inflammatory leucocytes or resident cells of the retina may promote the development and perpetuation of this condition.

The protective effects of omega-6 fatty acids in experimental autoimmune encephalomyelitis (EAE) in relation to transforming growth factor-beta 1 (TGF-β1) up-regulation and increased prostaglandin E2 (PGE2) production

Polyunsaturated fatty acids are known to affect the immune response and administration of the omega‐6 fatty acid linoleic acid has been reported to be beneficial in multiple sclerosis (MS) and EAE. In this study we have investigated the effects of oral feeding of plant lipid rich in the omega‐6 fatty acid gamma‐linolenic acid from Borago officinalis on acute and relapse disease and the immune response in EAE using SJL mice. EAE was induced by an encephalitogenic peptide (92–106) of myelin oligodendrocyte glycoprotein (MOG), and mice were fed the plant lipid daily from 7 days after EAE induction to assess the effects on acute disease and from day 25 to assess the effects on disease relapse. The clinical incidence and histological manifestations of acute EAE, and the clinical relapse phase of chronic relapsing EAE (CREAE) were markedly inhibited by omega‐6 fatty acid feeding. A significant increase in the production of TGF‐β1 in response to concanavalin A (Con A) at day 13 and a significant increase in TGF‐β1 and PGE2 to Con A, PPD and MOG peptide (92–106) at day 21 were detected in spleen mononuclear cells from fatty acid‐fed mice. There was no difference in interferon‐gamma, IL‐4 and IL‐2 production between the fatty acid‐fed and control groups. Significantly higher TGF‐β mRNA expression was found in the spleens of omega‐6 fatty acid‐fed mice at day 21. There were no differences in spleen cell proliferative response to Con A, PPD and MOG peptide (92–106). Biochemical analysis of spleen cell membrane fatty acids revealed significant increases in the eicosanoid precursor fatty acids dihomo‐γ‐linolenic acid and arachidonic acid in response to gamma‐linolenic acid feeding, indicating rapid metabolism to longer chain omega‐6 fatty acids. These results show that oral feeding of gamma‐linolenic acid‐rich plant lipid markedly affects the disease course of acute EAE and CREAE and is associated with an increase in cell membrane long chain omega‐6 fatty acids, production of PGE2 and gene transcription and, on activation, secretion of TGF‐β1.

ANTI-RETINAL AUTOIMMUNITY AND CIRCULATING IMMUNE COMPLEXES IN PATIENTS WITH RETINAL VASCULITIS

Sera from 44 patients with isolated retinal vasculitis (RV), 38 patients with retinal vasculitis accompanying systemic inflammatory diseases (RV + SID), and 33 patients with a similar range of systemic inflammatory diseases without eye involvement (SID alone) were assayed for circulating immune complexes (CIC) and for anti-retinal autoantibodies. CIC were present in 41% of patients with isolated RV and 55% of patients with RV + SID, whilst anti-retinal antibodies were present in about 70% of all patients with RV. 42% of those with SID alone had CIC and 30% of those with SID alone had retinal autoantibodies. Titres of anti-retinal antibodies were higher in patients with RV than in those with SID alone. In isolated RV there was an inverse relation between pronounced retinal autoimmunity and the occurrence of CIC--i.e., the more severe autoimmune retinal disease occurred in CIC-negative patients. Most patients with RV + SID tended to have mild or moderate retinal disease accompanied by both retinal autoantibodies and CIC, but severe retinal disease occurred in CIC-positive patients who did not have circulating anti-retinal antibodies. Patients with SID alone had high titres of retinal antibodies only when they were CIC-positive. It is suggested that the formation of CIC, possibly of an idiotype/anti-idiotype nature, may be a compensatory mechanism accompanying anti-retinal autoimmunity and that an imbalance between autoimmunity and immune complex formation may be an important predisposing factor in the development of retinal inflammatory disease.

Human antiretinal antibodies in toxoplasma retinochoroiditis

BACKGROUND/AIMS Toxoplasma retinochoroiditis (TR) is an important cause of blindness and visual morbidity, affecting young adults. It has been postulated that some of the retinal damage observed in TR is due to antiretinal autoimmune mechanisms. METHODS Humoral antiretinal autoimmunity in TR was investigated by indirect immunofluorescence (IIF) on normal human cadaveric retina and by a human retinal S-antigen ELISA. 36 patients with TR were separated on clinical grounds into those with first recurrence of disease (n=18) or those with multiple recurrences (n=18). Patients were also segregated into those with active (n=28) or quiescent disease (n=8). Serum from 16 normal controls (six with positive toxoplasma serology and 10 without) with no evidence of eye disease and 12 patients with idiopathic retinal vasculitis (IRV) were also tested. RESULTS Sera from 34 of the 36 patients (94%) with TR demonstrated photoreceptor layer reactivity by IIF contrasting with six of 16 normal controls (p= <0.001) and three of 12 IRV patients (p= <0.001). Titres of antiphotoreceptor antibody were also higher among TR patients than controls. Sera from 27 of the 36 TR patients, 10 of 16 normals, and nine of 12 retinal vasculitis patients possessed anti-human retinal S-antigen antibodies at a titre of 1:400 or more as assessed by ELISA (p= >0.05). Antiretinal autoantibody as detected by IIF did not run in parallel with S-antigen reactivity. CONCLUSIONS The data indicate that the extent of antiretinal reactivity within TR is not accounted for by anti-S-antigen antibodies alone. This remarkably high prevalence of antiphotoreceptor antibody in TR as opposed to that found in either healthy or disease controls suggest that these antibodies may be co-pathogenic in toxoplasma retinochoroiditis.

Human blood dendritic cells: binding to vascular endothelium and expression of adhesion molecules

To investigate the binding properties of dendritic cells (DC) to vascular endothelium, a comparative analysis was undertaken of DC, monocytes and lymphocytes isolated from the blood of 25 healthy subjects using monolayers of human umbilical vein endothelial cells as the adherence substrate. More blood DC (mean 24% adherence) were adherent to endothelial monolayers than monocytes (mean 18%; P < 0.001) and lymphocytes (mean 12%; P < 0.001). When the monolayers were pretreated with tumour necrosis factor‐alpha (TNF‐α) all leucocyte populations exhibited an increased attachment, but there was still greater binding of DC (mean 37% adherence) in comparison with monocytes (mean 23%; P < 0.001) and lymphocytes (mean 18%; P < 0.001). Flow cytometric analysis revealed that in relation to monocytes and lymphocytes the DC had a higher surface expression of the adhesion molecules CD11a (P < 0.05), CD11c (P < 0.05) and CD54 (P < 0.05) but a lower prevalence of cells bearing CD49d (mean 38%; P < 0.05) and the homing receptor CD62L (mean 14%; P < 0.001). CD1a was present on 22% of DC and virtually absent from the surface of monocytes and lymphocytes. The intensity of expression of the β1‐integrins, CD49c, CD49d and CD49e was greater on DC than lymphocytes and monocytes (P < 0.05). Antibody blocking studies demonstrated that DC binding to untreated and TNF‐α‐treated endothelium was dependent upon the expression of CD11a, CD18 and CD49d, and the simultaneous application of anti‐CD18 and anti‐CD49d antibodies produced an approximate 70% inhibition of adhesion (P < 0.001). Thus, the expression of both β1‐ and β2‐integrins contributes to the adhesive interaction between DC and endothelium.

Expression of mRNA coding for TNFα, IL-1β and IL-6 by cells infiltrating retinal membranes

Abstract• Background: Cellular mechanisms of inflammation are thought to be involved in the pathogenesis of proliferative vitreoretinopathy, and cytokines, which are products of cell activation, are known to play an important role in the development and maintainance of inflammatory reactions. It was the aim of this work to investigate the presence of cells expressing cytokine mRNA within retinal membranes. • Methods The presence of mRNA coding for the cytokines interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumour necrosis factor α (TNFα) was investigated in 19 epiretinal membranes obtained from eyes undergoing vitrectomy for the treatment of retinal detachment complicated by proliferative vitreoretinopathy. • Results Cells expressing mRNA for IL-1β were observed in 7 membranes, cells positive for IL-6 mRNA were seen in 12 membranes, and cells exhibiting mRNA for TNFα were present in 9 specimens. Only three membranes contained cells expressing mRNA for all the cytokines investigated. Four membranes possessed positive cells for IL-6 and TNFα, two contained cells expressing mRNA for IL-6 and IL-1β, and two others exhibited cells expressing mRNA for TNFα and IL-1β. Five membranes contained IL-6 mRNA-positive cells only, whilst two exhibited cells expressing mRNA for IL-1β, or TNFα only. • Conclusion The present findings indicate that cellular activation may occur during the development of PVR, and suggest that these cytokines may be locally produced by cells infiltrating epiretinal membranes. The presence of IL-1β, IL-6 and TNFα mRNA-positive cells within retinal membranes provides further evidence of a pathogenic role of these cytokines in proliferative vitreoretinopathy.

Soluble intercellular adhesion molecule-l (sICAM-1) as a marker of disease relapse in idiopathic uveoretinitis

This study reports the results of a point prevalence study of markers of endothelial dysfunction in the serum of patients with idiopathic uveoretinitis. sICAM‐l, soluble endothelial leucocyte adhesion molecule (sELAM), anti‐endothelial cell antibodies (AECA) and von Willebrand factor (vWF) levels were measured in 32 patients with isolated idiopathic uveoretinitis and seven with uveitis in association with systemic disease, using commercial and in‐house ELISAs. Raised levels of AECA were found in 31% of patients with isolated uveitis, vWFin 28%, sELAM in 15‐6% and sICAM‐1 in 31%. Further analysis revealed that raised sICAM‐1 levels were closely associated with recent relapse of disease (P=000003). Patients with accompanying systemic disease were found to have a similar prevalence of these serum abnormalities to those with isolated ocular disease. In conclusion, vascular endothelial dysfunction may contribute to pathogenesis in uveoretinitis, and in particular sICAM‐1 may prove a marker of disease relapse in this condition.