CYTOKINE AND CHEMOKINE EXPRESSION KINETICS AFTER CORNEAL TRANSPLANTATION¹

Abstract

Background. 

Allogeneic rejection is the most common cause of corneal graft failure. The aim of this work was to establish the kinetics of cytokine and chemokine mRNA expression before and after onset of corneal graft rejection.

Methods. 

Intracorneal cytokine and chemokine mRNA levels were investigated in the Brown Norway→Lewis inbred rat model in which rejection onset is observed at 8/9 days after grafting in all animals. Nongrafted corneas and syngeneic (Lewis→Lewis) corneal transplants were used as controls. Donor and recipient cornea was examined by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) for hypoxyanthine phosphoribosyltransferase (HPRT), CD3, CD25, interleukin (IL)-1β, IL-1RA, IL-2, IL-6, IL-10, interferon-γ (IFN-γ), tumor necrosis factor (TNF), transforming growth factor (TGF)-β1, and macrophage inflammatory protein (MIP)-II and by nonquantitative RT-PCR for IL-4, IL-5, IL-12 p40, IL-13, TGF-β2, monocyte chemotactic protein-1 (MCP-1), MIP-1α, MIP-1β, and RANTES (for regulated upon activation normal T cell expressed and secreted).

Results. 

A biphasic expression of cytokine and chemokine mRNA was found after transplantation. During the early phase (days 3–9), there was an elevation of the majority of the cytokines examined, including IL-1β, IL-6, IL-10, IL-12 p40, and MIP-II. There was no difference in cytokine expression patterns between allogeneic or syngeneic recipients at this time. In syngeneic recipients, cytokine levels reduced to pretransplant levels by day 13, whereas levels of all cytokines rose after observed rejection onset in the allografts, including TGF-β1, TGF-β2, and IL-1RA. The T cell-derived cytokines IL-4, IL-13, and IFN-γ were detected only during the rejection phase in allogeneic recipients.

Conclusions. 

There is an early cytokine and chemokine response to the transplantation process, evident in syngeneic and allogeneic grafts, that probably drives angiogenesis, leukocyte recruitment, and affects leukocyte functions. After an immune response has been generated, allogeneic rejection results in the expression of Th1 cytokines (IL-2, IL-12 p40, IFN-γ), Th2 cytokines (IL-4, IL-6, IL-10, and IL-13), and anti-inflammatory/Th3 cytokines (TGF-β1/2 and IL-1RA).

Unlike grafts of vascularized organs, avascular human corneal allografts survive indefinitely in the majority of patients without the need for systemic immunosuppressive therapy or HLA tissue matching. Corneal allograft rejection, however, is a major complication after transplantation in patients with rejected previous grafts and/or a vascularized recipient cornea. The healthy cornea is transparent because of the ordered array of stromal collagen fibers and the pump function of its endothelial cell monolayer. Immune responses within the cornea have potential to disturb stromal microanatomy or damage the nonreplicative endothelium, which is essential for clarity. The eye has a range of mechanisms, anatomical, physiological, and immunological, that are designed to prevent or at least limit tissue-damaging immune responses (¹). Tissue-damaging immune responses such as corneal graft rejection can still occur but only once these mechanisms have been overcome (²).

The cornea is composed of three distinct cellular compartments. The outer corneal epithelium consists of five or six layers of epithelial cells. The relatively acellular collagenous stroma contains some modified fibroblasts (keratocytes). A single layer of corneal endothelial cells exposed to the aqueous humor form the inner surface. The normal cornea is almost devoid of MHC class II antigen-presenting cells (APCs), other than the peripheral corneal epithelium, which contains a small population of Langerhans cells (LCs). This relative exclusion of LCs from the central region of the cornea contributes considerably to immune privilege (³). The resident epithelial, endothelial, keratocyte, and Langerhans cells within the cornea can all produce cytokines and chemokines both constitutively and after stimulation. Cultured corneal endothelial cells constitutively express interleukin (IL)-1α and transforming growth factor (TGF)-β1/2 (⁴). Corneal epithelial cells and keratocytes constitutively produce IL-1RA mRNA and protein (⁵). Cultured keratocytes can produce IL-1α/β, IL-6, IL-8, and tumor necrosis factor (TNF) upon stimulation by UV light (⁶) and monocyte chemotactic protein-1 (MCP-1) and regulated upon activation normal T cell expressed and secreted (RANTES) after stimulation with TNF/IL-1α (⁷). Human corneal endothelial cells and keratocytes produce low levels of IL-8 constitutively (⁸). IL-1 and TNF stimulate its production in keratocytes, epithelial, and endothelial cells (^{8, 9}).

The cell infiltrate during corneal allograft rejection in a rabbit model is a heterogeneous population consisting of macrophages, lymphocytes, plasma cells, and neutrophils (^{10, 11}). Fifty to 70% of the leukocytes are T lymphocytes, however, with only 10–30% expressing myeloid markers (¹²). Similar observations have been found in rat models where CD4⁺, CD8⁺, and CD25⁺ T cells have been identified in graft infiltrates and within the aqueous humor during rejection (^{13, 14}). Antibodies to CD4 can promote graft survival in both mouse (¹⁵) and rat (¹⁶) models. Evidence, therefore, strongly suggests that T cells predominantly mediate graft rejection, in these animal models.

The presence of Th1-type cytokines and the comparative paucity of Th2-type cytokines both at the mRNA (¹⁷) and protein level (^{18, 19}) during corneal graft rejection imply that graft rejection is mediated by Th1-type T lymphocytes. The aim of these experiments was to further characterize the cytokine profile and by inference the cell populations that mediate graft rejection with the aim of developing better-targeted immune intervention.

MATERIALS AND METHODS

Animals.

Inbred female Lewis (RT1^l) and Brown Norway (BN, RT1ⁿ) strain rats (200–250 g) were obtained from Harlan Olac UK (Bicester, England). Lewis rats were used as recipients and syngeneic graft donors. Allograft donors were BN strain. All animals were allowed unrestricted access to food and water. United Kingdom Home Office regulations for care of experimental animals were observed at all times.

Corneal transplantation.

A 3.5-mm diameter full thickness corneal graft was trephined from the center of the donor cornea and transplanted into a 3.0-mm diameter recipient corneal bed. The surgical technique used for penetrating keratoplasty was similar to that described by Williams and Coster (²⁰), and all rats received a unilateral graft. Chloramphenicol ointment was administered to the recipient eye immediately after surgery. No immunosuppressive agent was administered at any time. Sutures were removed on day 7 after transplantation.

Assessment of grafts.

Rats with surgical complications (cataract or anterior synechiae), which might prejudice graft outcome or confuse diagnosis of graft rejection were excluded from the study. Corneal transplants were scored using established visual criteria for corneal opacity (0–4), edema (0–2), and vascularization (0–4) (¹⁴). Graft rejection was diagnosed on the day on which, in a previously clear graft, a score of 5 was reached and with an opacity grade of at least 3 in all cases. Using this assessment system, rejection onset was observed in BN→Lewis allografts between days 8 and 9 after transplant and Lewis→Lewis syngenic grafts showed no signs of rejection.

RNA isolation and reverse transcription.

Donor and the surrounding recipient cornea were excised on days 3, 5, 7, 9, 11, and 13 after transplantation, dissected apart, snap-frozen in liquid nitrogen, and stored at −70°C. Corneal tissue removed from ungrafted Lewis and BN animals acted as controls (day 0). After homogenization of the cornea using a 0.5-ml micro tissue grinder (Wheaton Science Products), cellular mRNA was extracted using RNAzol™ B (AMS Biotechnology Ltd, Oxon, UK). The mRNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (GIBCO BRL) in the reverse transcriptase buffer and incubated at 37°C for 40 min. Another 2 μl of Moloney murine leukemia virus reverse transcriptase was added and incubated for another 40 min at 37°C. To stop the reaction, the samples were heated at 70°C for 10 min.

Reverse transcription-polymerase chain reaction (RT-PCR).

All PCR reactions were initially optimized (for annealing temperature and MgCl₂ concentration) using rat spleen cells stimulated with phytohemagglutinin (1 μg/ml; Sigma Chemical, St. Louis, MO) and phorbol myristate acetate (10 ng/ml; Sigma Chemical) (18 hr). With the exception of hypoxyanthine phosphoribosyltransferase (HPRT; 30 cycles) and IL-4 (40 cycles), all PCR reactions were carried out for 35 cycles using an Omnigene Thermal cycler (Hybaid, London, UK). Each 20-μl PCR reaction consisted of: 0.5 U of Taq polymerase (Qiagen GmbH, Hilden, Germany), 1× PCR reaction buffer, 0.2 mM of each dNTP (Pharmacia, Milton Keynes, UK), 1 μM of each primer, 5 μl of diluted cornea cDNA (each recipient/donor cornea cDNA sample was diluted to a total volume of 375 μl) and 5 μl of competitor plasmid. The PCR reaction was hot-started at 94°C for 2 min to denature all cDNA samples.

Primer sequences.

All primers were obtained from MWG-Biotech, Milton Keynes, UK. All sequences have been published previously: HPRT, CD3, CD25, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, macrophage inflammatory protein (MIP)-2, and interferon-γ (IFN-γ) (²¹); IL-1RA, TGF-β1, TNF, and MCP-1 (²²); RANTES, MIP-1α, and MIP-1β (²³); IL-12 p40 (²⁴); IL-13 (²⁵); and TGF-β2 (²⁶).

Competitive RT-PCR.

Competitive RT-PCR was carried out using methodology previously described (^{21, 22, 27–29}). Briefly, PCR reactions were carried out in six replicate tubes containing different concentrations of competitor plasmid DNA (kind gifts of Dr. Volk [²¹] and Dr. Shire [²²]) (6×10-fold dilutions starting with 25 pg/ml). Ten microliters of PCR product was visualized by agarose gel electrophoresis and ethidium bromide staining, and analyzed using a Gel Doc 1000 video gel documentation system (BioRad, Hemel Hempstead, UK). The intensity of each band was calculated using multianalyst software (BioRad). Several macros were generated within Excel (Microsoft, Redmond, WA) to convert these data to obtain the point of equivalence (i.e., the point at which the Taq polymerase has amplified an equivalent amount of both the wild-type product and the competitor plasmid product). All data are expressed as the relative point of equivalence (moles of competitor plasmid).

Noncompetitive RT-PCR.

We assayed for the expression of the cytokines IL-4, IL-5, IL-12 p40, IL-13, and TGF-β2 and the chemokines MIP-1α, MIP-1β, RANTES, and MCP-1 using noncompetitive RT-PCR. This method does not give a quantitative measure of mRNA levels. However, on a background of minimal changes in housekeeping gene expression (HPRT, Fig. 1a), noncompetitive RT-PCR is sufficient to detect gross changes in cytokine and chemokine mRNA expression.

Figure 1: (a–d) Expression levels of mRNA for HPRT (a), CD3 (b), CD25 (c), and IL-2 (d) from peripheral and central cornea samples after transplantation, days 3–13 (•, allogeneic; ○, syngeneic), and untransplanted cornea, day 0 (♦, Brown Norway; ⋄, Lewis). Messenger RNA concentration is expressed as picomoles of competitor sample (at the point of equivalence, see Materials and Methods)/corneal sample. (e–h) Expression levels of mRNA for IL-1RA (e), IL-1β (f), IL-6 (g), and IL-10 (h) from peripheral and central cornea samples after transplantation, days 3–13 (•, allogeneic; ○, syngeneic), and untransplanted cornea, day 0 (♦, Brown Norway; ⋄, Lewis). Messenger RNA concentration is expressed as picomoles of competitor sample (at the point of equivalence, see Materials and Methods)/corneal sample. (i-l) Expression levels of mRNA for TGF-β1(i), MIP-II (j), TNF (k), and IFN-γ (l) from peripheral and central cornea samples after transplantation, days 3–13 (•, allogeneic; ○, syngeneic), and untransplanted cornea, day 0 (♦, Brown Norway; ⋄, Lewis). Messenger RNA concentration is expressed as picomoles of competitor sample (at the point of equivalence, see Materials and Methods)/corneal sample.

Figure 1: Continued

Figure 1: Continued

RESULTS

HPRT mRNA.

In all experiments, the mRNA levels are compared from central cornea (derived from the donor) and peripheral cornea (derived from the recipient). There were only small differences in HPRT mRNA levels between different samples (Fig. 1a). Data were not normalized to HPRT levels, however, inasmuch as, coincident with the influx of inflammatory cells, there was a small but marked increase in HPRT mRNA after rejection.

T-cell markers.

Increased levels of mRNA encoding CD3 and CD25 were found after transplantation of both allogeneic and syngeneic corneas, rising to higher levels in allografts at days 9–13 (Fig. 1, b and c).

T cell-derived cytokines.

mRNA for the T cell-specific effector cytokines IFN-γ, IL-4, and IL-13 were only detected in allografts after rejection (days 9–13). mRNA for these cytokines were not detected in any syngeneic samples (Figs. 1l and 2). Low levels of IL-2 mRNA were detected, particularly within the peripheral cornea of both allogeneic and syngeneic recipients during the first 7 days after grafting. Expression levels rose, within both recipient and donor tissue, upon rejection (Fig. 1d). Of interest, we found IL-2 mRNA expression from normal rat corneas of several inbred strains (LOU, Lewis, BN, PVG, AO, AUG, and WAG) (unpublished data), with higher expression in the peripheral than the central cornea in all strains. Detection of IL-2 within corneal resident cells has implications for immune privilege, as Fas ligand-mediated activation-induced cell death is dependent on the presence of exogenous IL-2 (³⁰). IL-2 transcripts detected with the sensitive PCR method have not, however, been confirmed at the protein level (¹⁸), suggesting that functional IL-2 protein is not present within normal cornea.

Figure 2: Results of RT-PCR for the chemokines RANTES, MCP-1, MIP-1α, and MIP-1β and the cytokines TGF-β2, IL-13, IL-12 (p40), IL-4, and IL-5. Donor cornea was used for all PCR reactions with the exception of IL-4 and IL-5, where recipient cornea was used. All PCRs were carried out for 35 cycles with exception of IL-4 (40 cycles). Phytohemagglutinin/phorbol myristate acetate-stimulated rat spleen cells were used as the positive control, except for TGF-β2 where normal rat cornea was used. Negative control consists of all PCR reagents being used in the absence of cDNA.

Early response cytokines.

IL-1β, IL-5, IL-6, IL-10, and IL-12 (p40) all share a similar profile of expression. These cytokines were either absent or expressed at low levels in normal corneas, and were up-regulated immediately after transplantation in both allogeneic and syngeneic recipients. The expression levels then continued to rise in allogeneic recipients upon rejection or decline back to normal levels in syngeneic samples. Early expression of these cytokines is more likely to mediate a generalized response to tissue injury, rather than an allogeneic response. TNF mRNA was detected at low levels at all time points with higher expression upon rejection in allogeneic recipients (Fig. 1k). We have previously detected high levels of bioactive TNF protein in the aqueous humor before and during graft rejection in a rabbit model (unpublished data). As a range of posttranslational mechanisms tightly regulates the bioactivity of TNF, mRNA expression levels may not correlate well with levels of functional protein.

Anti-inflammatory proteins (TGF-β1/2/IL-1RA).

Normal cornea constitutively expresses mRNA encoding the anti-inflammatory proteins IL-1RA and TGF-β1/2. mRNA levels remain high after transplantation with a 5–10-fold increase of expression upon rejection in allograft recipients (Fig. 1e,i and Fig. 2).

Chemokine expression.

MIP-II is a murine/rat CXC chemokine homolog of human IL-8 and functions as a neutrophil chemoattractant. It contains the ELR motif and is capable of inducing angiogenesis. Although mRNA for MIP-II is present in normal cornea, it was up-regulated after transplantation, with peak expression at day 5, probably contributing to blood and lymph vessel induction seen at this time. Levels continued to fall in syngeneic recipients, reaching background levels by day 13. A second peak of expression was detected in allogeneic recipients during rejection (Fig. 1j). A similar pattern of expression was also seen for the CC chemokines RANTES, MCP-1, MIP-1α, and MIP-1β using nonquantitative PCR methods (Fig. 2).

Donor/recipient cytokine expression.

Profiles of cytokine expression were almost identical between central (donor) and peripheral (recipient) corneal samples, particularly in the period before rejection onset, as has been found previously (¹⁷). Slight differences were detected during rejection at days 11 and 13 where 5–10-fold higher levels of expression of most cytokines (in particularly IL-10, MIP-II, and IFN-γ) were detected within donor cornea compared to recipient cornea. This observation is in keeping with the concept of a generalized inflammatory response to the transplantation procedure during the early immune response (similar levels in donor and recipient samples), followed by an alloantigen-specific immune response occurring during rejection (indicated by restriction of expression to donor cornea). Both IL-10 and MIP-II have angiogenic properties; therefore, it is not surprising that their expression would be specific to the site of inflammation. IL-10 is also released after induction of apoptosis of activated T cells via the Fas-Fas ligand pathway, a process that is probably taking place as donor-specific activated T cells come into contact with donor endothelial cells expressing Fas (^{31, 32}).

DISCUSSION

Cytokine and chemokine expression during corneal transplant rejection has been studied previously both at the mRNA level (¹⁷) and at the protein level (^{18, 19}). In this study, we have used competitive RT-PCR to measure cytokine and chemokine mRNA expression levels. Although other technologies including fluorescent-based approaches can be used, this is a validated method for measuring mRNA levels (^{21, 22, 27–29}). For some cytokines and chemokines, we have used noncompetitive RT-PCR, which can detect gross changes in mRNA levels. The cytokine and chemokine data presented here extends previous studies both in regard to the panel of cytokines and chemokines examined, and also by providing a quantitative analysis of mRNA expression (summarized in Table 1). There is an early peak of mRNA expression between days 3 and 7 of the cytokines IL-1β, IL-5, IL-6, IL-10, IL-12 (p40), the CXC chemokine MIP-II and the CC chemokines RANTES, MIP-1α, MIP-1β, and MCP-1. This early response is seen in both syngeneic and allogeneic recipients, with expression levels being almost identical between the two groups. In syngeneic recipients, there was vascularization of the recipient cornea to the graft-host junction until day 7, and the cytokine mRNA expression levels decreased after this time. In allogeneic recipients, a second peak of cytokine mRNA expression was seen coincident with the observed onset of graft rejection. This second peak of cytokine mRNA expression includes all the cytokines present in the initial peak, but also contains the T cell-derived effector cytokines IL-4, IL-13, and IFN-γ, which were not detected before rejection in allogeneic recipients or in syngeneic recipients at any time.

Table 1: Summary of cytokine expression levels for quatitative RT-PCR and estimated mRNA expression levels for the non-quantitative RT-PCR results^a

Previous experiments have shown that the mere process of excising BALB/c mouse cornea is sufficient to stimulate rapid IL-1α secretion (³³). The early response that we observed between days 3 and 7 is probably a consequence of the surgical process (physical damage to the cornea and presence of suture material). It is largely independent of the antigenic phenotype of the donor cornea, as cytokine expression patterns during this time were almost identical in allo- and syngeneic grafts. IL-1α and TNF protein up-regulation was also detected at comparable levels in allogeneic and syngeneic graft recipients immediately after transplantation (¹⁸). Addition of IL-1 to corneas cultured in vitro has been shown to up-regulate expression of IL-6 (^{33, 34}), IL-8 (⁸), MCP-1, and RANTES (⁷). Therefore, a cascade of proinflammatory cytokine and chemokine expression is initiated from the surgical trauma, which probably mediates angiogenesis and subsequent migration and activation of appropriate inflammatory cells. Peripheral LCs have been shown to migrate to the central region of the cornea, which is APC-free, after intracorneal injection of TNF (³⁵), IL-1 (³), IL-2, IL-6, and IFN-γ (³⁶). Furthermore, centripetal migration of LCs, within 30 min, of intracorneal injection of IL-1 has been demonstrated, suggesting a direct LC chemotactic role for IL-1 (³).

We have confirmed the constitutive corneal expression of IL-1RA mRNA reported by others, and demonstrated that the high expression levels remain constant after transplantation. The proinflammatory properties of IL-1 are suppressed in the cornea by constitutive expression in the epithelium of IL-1RA (³⁷). Moreover, topical application of IL-1RA has been shown to suppress migration of LCs (³⁸) and promote corneal transplant survival (³⁹). IL-1β expression levels by comparison are dramatically up-regulated by day 3 after transplantation. This disruption in the balance of pro- and anti-inflammatory isoforms of IL-1 might have a role in induction of LC migration into the center of the cornea and promote the early immune response.

In contrast to the cornea, vascularized organ allografts destined to be rejected express higher levels of particular cytokines within 1–3 days of transplantation, compared to their accepted syngeneic controls. For example, early expression of mRNA encoding, IL-2, IL-4, and IFN-β in mouse cardiac transplants (⁴⁰) and IFN-γ, IL-2, IL-4, and IL-10 in rat hepatic and renal transplants (⁴¹) are early predictors of rejection. Interestingly, cytokine immune responses to other nonvascularized grafts, e.g., pancreatic islet cells, show similarities between allogeneic and syngeneic grafts at least for the first 4 days after transplantation. After day 4, there was an allogeneic-specific increase in cell infiltrate and cytokine expression (⁴²). Expression of T cell-derived cytokines as early as day 3 within vascularized organs is by cells recognizing alloantigen via the direct pathway. Because of the paucity of passenger leukocytes (LCs), within the donor cornea, allorecognition functions via the indirect pathway. Therefore, T cell-derived cytokines are only detected in the cornea after an interval during which donor antigen is processed and presented via the indirect pathway.

When APCs are exposed to antigen in the presence of TGF-β, which is constitutively expressed within the eye, they secrete IL-10 in preference to IL-12 (⁴³). Some antigens, however, are capable of inducing IL-12 expression by APCs even in the presence of TGF-β (for example, UV5C25 tumor cells). We have detected early up-regulation of IL-12 mRNA after transplantation in both syngeneic and allogeneic graft recipients, suggesting that the production of IL-12 from APCs after transplantation is nonspecific. This production could be a result of either the trauma and vascularization induced by transplantation or, alternatively, of the recruitment of nonresident, peripheral APCs not exposed to, or resistant, to the effects of TGF-β. In any case, this suggests that strategies directed against the early inflammatory response may be effective at maintaining the counter-inflammatory environment within the eye derived in part from TGF-β function.

In summary, we found increased mRNA expression of Th1-type cytokines (IL-2, IFN-γ, IL-12 p40), Th2-type cytokines (IL-4, IL-5, IL-6, IL-10, IL-13), and Th3/anti-inflammatory cytokines (TGF-β1/2 and IL-1RA) within the graft during rejection. An important limitation of this study is that we have not addressed whether the expression of mRNA leads to the production of functional cytokine proteins during rejection. Previous studies at both the mRNA and protein levels have suggested that Th1 T cells mediate corneal graft rejection (^17–19). Given the up-regulated expression of all cytokines examined, it is difficult to conclude from our data that only one T-cell phenotype is functional during rejection. One possible interpretation of our data is that corneal graft rejection is mediated by Th1-derived cytokines alone and that mRNA for Th2/Th3-type cytokines is derived from nonactivated, nonallospecific T cells. It is perhaps more likely that a fully mismatched allogeneic immune response involves activated allospecific T cells with a range of effector functions, having different roles during the allogeneic response. Th1 cytokines almost certainly are involved in the rejection process. Th2 cytokines, however, could conceivably contribute to protection and/or rejection.

A full understanding of the cytokines and chemokines produced during rejection may suggest future therapeutic strategies, including the expression of antagonistic molecules. These could be delivered using gene transfer approaches, currently being developed (^44–46). The early cytokine response seen in both allogeneic and syngeneic recipients contributes to vascularization, a loss of immune privilege and induction of an inflammatory response. Therapeutic strategies directed at blocking this response, maintaining the natural immune privilege of the cornea, may prove to be as effective as strategies that block the allo-immune response, through either blockade of costimulation or the IL-2 receptor.