BACKGROUND/AIMS Scientific interest in pentoxifylline has been reawakened owing to the recognised effects of this drug on immune functions, particularly its influence on cytokine production. In a previous study, the authors demonstrated that spiking of organ culture media with endotoxin elicited a marked enhancement in the release of IL-6 and IL-8 from corneal tissue and that these events coincided with degenerative changes in endothelial cells and a higher incidence of actual loss among this population. Since traces of donor derived endotoxin can be detected in up to 50% of corneal organ cultures, this substance may have a direct influence on graft viability or trigger inflammatory responses in the host. They, therefore, wished to ascertain whether supplementation of media with pentoxifylline improved endothelial cell survival in organ cultured donor corneas.
METHODS 12 fellow pairs of donor corneas were cultured for 20 days, with a change of medium on day 10: One of each pair was incubated in the absence, and the other in the presence, of pentoxifylline (25 μg/ml). Samples of medium were withdrawn at regular intervals during the course of incubation and screened for cytokines IL-6, IL-8, and prostaglandin E2 by ELISA. Endothelial cell morphology and numerical density were assessed on days 0, 10 and 20.
RESULTS Addition of pentoxifylline to organ culture media led to a significant improvement in endothelial cell survival. This drug also elicited a significant increase in the level of IL-6 and marginally suppressed that of IL-8 during the initial 10 day phase of incubation. During the second 10–20 day phase, the level of both IL-6 and IL-8 decreased significantly in the presence of pentoxifylline, the relation between these two cytokines being the inverse of that observed in the absence of the drug. No significant changes in the level of prostaglandin E2 were apparent.
CONCLUSION The addition of pentoxifylline to organ culture media leads, ultimately, to a suppression of IL-6 and IL-8 secretion by corneal tissue. The potentially damaging effects of these cytokines are thereby quelled, as evidenced by the improvement in endothelial cell survival.
- organ culture
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Pentoxifylline, a derivative of methylxanthine theobromine, acts as a phosphodiesterase inhibitor and thereby elevates the levels of cAMP. This drug, which is widely used in the treatment of peripheral vascular disorders, is claimed to improve microcirculation and tissue oxygenation by increasing the flexibility of erythrocytes and by reducing blood viscosity and platelet aggregation.
Interest in pentoxifylline has been recently reawakened because of its reported suppressive action on immune functions,1-3particularly on cytokine production.3-6 It has been shown to be beneficial in the treatment of endotoxaemia,7-12tumour induced cachexia,13 inflammatory bowel disease,1415 and AIDS,16 as well as in organ transplantation.17-21 Pentoxifylline probably acts primarily by inhibiting tumour necrosis factor,10121321-24 but other cytokines, such as IL-1β,1025 IL-2,26IL-6,710 IL-8,2728IL-10,51625 γ-interferon,29 and transforming growth factor β,30 vascular endothelial growth factor, as well as adhesion molecules,31 are also implicated. This agent additionally reduces leucocytosis and neutrophilia, and inhibits the phagocytic and microbicidal activities of monocytes, macrophages and neutrophils,123233 as well as degranulation in the latter.8 It also modulates fibrinolytic activity, both in vitro and in vivo,6 by suppressing tissue type plasminogen activator and its inhibitor.34
In a previous study, we demonstrated that spiking of organ culture media with endotoxin markedly enhanced the release of IL-6 and IL-8 from corneal tissue. These events coincided with degenerative changes and a higher incidence of death in endothelial cells.35Since traces of donor derived endotoxin can be detected in up to 50% of organ culture corneas,36 it may be directly implicated in graft viability or in the triggering of host inflammatory responses. The addition of pentoxifylline to such cultures could have a beneficial effect on the condition of the donor corneas or even on the clinical course after grafting.
We have already tested the potential toxic effects of pentoxifylline on cultured cells and defined its therapeutic range.37 In the present study, we wished to ascertain whether this drug improved endothelial cell survival when added to the medium bathing organ cultured donor corneas, and whether this putative effect was correlated with the release of various cytokines.
Materials and methods
EVALUATION OF DONOR CORNEAS AND ORGAN CULTURING CONDITIONS
Donor globes were removed within 50 hours of death and examined with a slit lamp. The eyes were disinfected with PVP-iodine, and corneoscleral buttons then excised microsurgically under laminar flow conditions. Tissue discs were immersed in MEM (Seromed), and the morphological status of the endothelium evaluated according to standard criteria by inverted phase contrast microscopy.38 Corneal buttons were then transferred to wells containing buffered saline (Alcon) to promote swelling of the intercellular spaces. The endothelium was photographed using a high resolution video camera, and the central numerical densities estimated on video prints using the fixed frame technique.
Corneal discs, maintained in a vertical position by means of a standard holding device (Storz; no 93-100), were cultured in 60 ml of medium (MEM supplemented with 2% fetal calf serum, glutamine, HEPES-buffer, antibiotics, and amphotericin B (all purchased from Seromed)) contained within conventional tissue culture flasks (Falcon; no 3013E): They were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide, with a change of medium after 10 days.
Endothelial cell numerical densities were determined before, and on days 10 and 20, of organ culturing.
EXPERIMENTAL SET UP
Corneal discs from the fellow eyes of 12 donors were evaluated in this investigation; the incidence of endothelial cell loss incurred after culturing being correlated with the presence of various cytokines released into the medium. One of each pair was incubated in the absence (right corneas), and the other (left corneas) in the presence, of pentoxifylline (25 μg/ml).
Samples of fluid were withdrawn for analysis between days 0 (baseline value and negative control) and 20. On day 10, the corneas were transferred to fresh medium, and culturing continued for a further 10 days under the conditions described above. Corneal discs derived from the fellow eyes of an additional five donors were cultured and analysed only between days 0 and 10.
Aliquots of medium were screened for cytokines IL-6, IL-8, and prostaglandin E2 according to the sandwich ELISA technique. The detection level for each cytokine was 30 pg/ml.
All fluid samples, withdrawn before day 10, were tested for the presence of donor derived endotoxins using the QCL-1000 Chromogenic Limulus Amoebocyte Lysate Assay (BioWhittaker), the lower level for a positive result being set at 0.600 endotoxin units/ml.
ELISA results and endothelial cell numerical densities were compared statistically using Student's ttest.
The data distribution was evaluated using the Kolmogorov-Smirnov distance test.
The intereye variability was normal (p >0.20).
All donor corneas lost a significant number of endothelial cells during the organ culture period (p <0.001 (Fig 1)), but these losses (2–30%) lay well within the range to be expected under such conditions (Table 1).
Samples of medium withdrawn during the initial 10 day phase of incubation registered positive for endotoxin (level ⩾0.600 units/ml) in 20 of 34 cultures (Table 2).
Supplementation of media with pentoxifylline (25 μg/ml) led to a significant improvement in endothelial cell survival at the end of the 20 day organ culture period (p = 0.025 (Fig 1)). The introduction of this drug was also accompanied by a significant increase in the level of IL-6 secreted by donor corneas during the initial 10 day phase of organ culture (p ⩽0.001 (Fig 2)). No significant change in the level of either IL-8 or prostaglandin E2 was observed, although that of IL-8 lay below that of IL-6 in 11/17 instances.
In the absence of pentoxifylline, the level of IL-6 was significantly lower than that of IL-8 (p = 0.017 (Fig 2)).
During the second phase of organ culture (days 10–20), after the change of medium, pentoxifylline (25 μg/ml) induced a significant decrease in the levels of both IL-6 (p = 0.007) and IL-8 (p = 0.003 (Fig 3)), that of the latter being significantly lower than that of the former (p = 0.041 (Fig 3)).
No significant change in the level of prostaglandin E2 was invoked.
In the absence of pentoxifylline, the level of IL-6 tended to be somewhat lower than that of IL-8, also during this second phase of incubation, but the difference was not significant.
Pentoxifylline elicited no changes in the morphology of corneal endothelia.
Inflammatory reactions, whether of extraocular or intraocular origin, tend to be downregulated by the avascular, and hence immune privileged, cornea. This feature is recognised as being essential for a favourable graft prognosis. In situ, corneal tissue is protected from potential inflammatory stimulants, such as endotoxins, by the epithelial and vascular barriers; but in vitro, the situation is different. Under these conditions, contamination of culture media with endotoxins could have untoward effects: these may be manifested either directly, by overt changes in cell morphology, and/or by a more discrete influence on the tissue's immunological status, the consequences of which may be realised only after transplantation, by a compromised graft prognosis.
In the current study, we screened the media of donor corneas cultured in the absence or presence of pentoxifylline for various cytokine mediators of the inflammatory response and compared these data with findings of endothelial cell morphology and numerical density.
The addition of pentoxifylline to culture media elicited a significant increase in the release of IL-6 by donor corneas during the initial 10 day phase of incubation and a significant decrease during the second 10–20 day phase. In contrast, the release of IL-8 from corneal tissue was marginally suppressed during the initial phase of incubation but decreased significantly during the second.
The relation between the levels of IL-6 and IL-8 also changed during the two phases of incubation. During the initial period no significant difference was observed between the levels of IL-6 and IL-8, but after the change of medium, the level of IL-8 was significantly lower than that of IL-6. The relation manifested during the second phase of incubation represents an inversion of that observed under standard conditions in the absence of pentoxifylline.
Pentoxifylline also elicited a significant improvement in endothelial cell survival at the end of the culturing period.
As previous studies have revealed, the levels of IL-6 and IL-8 under standard conditions and after endotoxin stimulation are highest during the initial 10 day phase of incubation,35 when the autocrine or paracrine functions of responsive cells intrinsic to the cornea or belonging to the passenger pool (for example, macrophages, Langerhans cells, or lymphocytes) are still operative. But during the change of medium, at least a portion of the responsive cells may be readily dislodged and washed away. Alternatively, the susceptible cell population may dislodged and washed away or become refractory.35
IL-6 is expressed by a number of cell types, and has an important role in host defence and immune responses. It regulates T lymphocyte function, and enhances the expression of Fc receptors on macrophages as well as stimulates their phagocytic activity. In endotoxin induced uveitis, IL-6 has been shown to elicit more damage than endotoxin itself when injected intravitreally, and is known to increase the expression of HLA class II antigens, a finding which may be of particular relevance in corneal grafting.39
The secretion of IL-8 by organ cultured corneas may play a part in directing the migration of leucocytes towards this tissue from neighbouring ones. Infiltration of the cornea with such cells is a characteristic of both infectious and non-infectious ocular inflammation, including keratitis, uveitis, and allograft rejection.40 And the production of IL-8 by corneal tissue in culture may thus have important clinical consequences.
Since up to 50% of organ culture media may contain endotoxins, in spite of their being sterile,36 instances of endothelial cell death may be partially attributable to these substances being carried over with excised tissue and their subsequent stimulation of cytokine production. These latter mediators of inflammation may, in turn, elicit an increased expression of HLA class II antigens, which, by upregulating the recipient's local immune response, may lead to graft rejection and failure.
This study has demonstrated that when organ culture media are supplemented with pentoxifylline, these deleterious effects may be eschewed, as evidenced by the improved survival of endothelial cells. Hence, the routine addition of this drug to organ culture media could have a beneficial influence not only on the condition of the donor corneas but also on their clinical course after grafting. It should be borne in mind, however, that the half life of pentoxifylline in organ culture media has not yet been determined and that the techniques currently implemented for measuring donor derived endotoxin cannot distinguish between small quantities of this substance with high activity and large quantities with low activity. The clinical course of pentoxifylline supplemented organ cultured donor corneas also needs to be evaluated before we can draw any definitive conclusions.
The authors would like to express their appreciation to Dr C England for her assistance in preparation of the manuscript and to Ms F Flückiger and Ms M Gygax for their technical assistance.
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