Heterogeneous Induction of Major Histocompatibility Complex Class II Antigens on Corneal Endothelium by Lnterferon-7

Heterogeneous Induction of Major Histocompatibility Complex Class II Antigens on Corneal Endothelium by lnterferon-7

Beafnjs J. J. Foers,* Joosr J. von den Oord,t Alfons Billiau,}: Jo Von Damme,:}: and Luc Missotren*

The expression and distribution of major histocompatibility complex (MHC) class II gene products, HLA-DR, HLA-DQ, and the HLA-DR invariant chain, were studied on flat mounts of human corneal endothelial cells (HCEC) after in vitro incubation of donor corneas with interferon-gamma (IFN-7), interleukin-1 (IL-1), and IL-6, using a sensitive immunoperoxidase technique with monoclonal antibodies. Control HCEC and endothelium treated with IL-1 or IL-6 completely lacked MHC class II antigens. After treatment with 50 U/ml, 100 U/ml, 500 U/ml, and 5000 U/ml of human IFN-7, a mosaic-like, patchy staining for all MHC class II products was observed: part of the HCEC showed membranous and/or cytoplasmic positivity; other endothelial cells were negative. In addition, a dose- dependent response to IFN-7 was observed: the proportion of cells expressing class II products rose with increasing doses of IFN-7. The induction of MHC class II antigen expression on HCEC by IFN-7 was completely inhibited by the addition of a neutralizing antibody directed to IFN-7 but not by IL-1/?. The significance of these findings with respect to corneal transplantation immunology is discussed. Invest Ophthalmol Vis Sci 32:341-345, 1991

The corneal endothelium is a major target in im- vestigated the effect of various doses of IFN-7, inter- mune rejection of corneal allografts. Although prod- leukin-1 (IL-1), and IL-6 on the induction of MHC ucts encoded by the major histocompatibility com- class II products in human corneal endothelial cells plex (MHC) play a key role in the initiation and (HCEC). Our results indicate that these cells are maintenance of rejection processes, the endothelial strikingly heterogeneous with respect to their re- cells of normal, freshly excised adult corneas are de- sponse to IFN-7. void of both MHC class I (or HLA-A, -B, and -C) and class II (or HLA-DR) antigens.1"3 Materials and Methods De novo appearance of MHC products has been Eight cadaver corneas were obtained from the reported in the endothelium of rejected corneal but- 8 4 5 Leuven Eye Bank. These corneas were excised from tons ' ; moreover, in vitro experiments have shown human eyes removed post mortem and stored for that MHC class II antigens can be induced on cul- various intervals in Minimum Essential Medium tured corneal endothelial cells by interferon-7 6 7 (Gibco, Paisly, Scotland) supplemented with 10% (IFN-7). ' However, neither the exact distribution of fetal calf serum (Gibco), 50 U/ml nystatin, 0.1 mg/ml the responding endothelial cells, nor the effect of amikacin, and 0.5 mg/ml ticarcillin. One additional other cytokines, are known as yet. Making use of the cornea was obtained from an eye that was operatively en face mounting technique of corneal endothelial removed because of primary malignant melanoma of cell-Descemet membrane complexes and a panel of the choroid. The viability of the HCEC layer was well-characterized monoclonal antibodies directed to evaluated by vital staining with Evans' blue. MHC class II products and related antigens, we in- Escherichia co//-derived human IFN-7 was cour- teously provided by Dr. W. W. Wolf (Bioferon, Lau- From the *Department of Ophthalmology, fDepartment of Pa- pheim, Germany). The IL-1/3 and IL-6 were derived thology, Laboratory of Histo- and Cytochemistry, tRega Institute from human peripheral blood leukocytes and were 9 for Medical Microbiology, University of Leuven, B-3000 Leuven, purified to homogeneity. D9D10 is a neutralizing Belgium. monoclonal antibody against human IFN-7.10 Submitted for publication: February 13, 1990; accepted Sep- The corneal fragments were incubated with cyto- tember 24, 1990. Reprint requests: B. J. J. Foets, Department of Ophthalmology, kines for 4 days at 37°C. In all experiments, a frag- University Hospital Sint-Rafael, Kapucijnenvoer 33, B-3000 Leu- ment that did not receive cytokines, served as a con- ven, Belgium. trol. Subsequently, the Descemet's membrane with

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the covering endothelial cell layer1' was gently peeled 1G-H). In general, the number of HCEC expressing off from the underlying stroma using a fine, monofil- the HLA-DR invariant or 7-chain outnumbered ament forceps. The samples of Descemet's mem- those expressing a- or /^-chains. brane-endothelium complex were mounted on albu- To exclude nonspecific induction of MHC class II min-coated slides, dried at room temperature, and products by contaminants in the recombinant IFN-7, fixed for 10 min in absolute acetone. the effect of addition of a neutralizing monoclonal The MHC class II products and related antigens antibody D9D10 directed to IFN-7 was studied. For were detected with a three-step avidin-biotin-peroxi- this purpose, small parts of two cadaver corneas were dase complex (ABC) method. Rehydrated slides were incubated in standard culture medium, in medium incubated with the following monoclonal antibodies: supplemented with IFN-7 (100 or 500 U/ml), in me- TAL1B5 (Imperial Cancer Research Fund, London, dium containing the same doses of IFN-7 to which UK) and L243 (Becton-Dickinson, Mechelen, Bel- anti-IFN-7 antibody was added in excess before the gium), directed to the monomorphic part of HLA- time of incubation, or in medium containing the an- DR a- and /3-chains respectively; LN3 (Biotest, Brus- tibody alone. sels, Belgium) and CR3/43 (DY Mason, Oxford, The induction of MHC class II product expression UK), directed to nonpolymorphic parts of HLA-DR was completely inhibited by the addition of antibody, antigens; LN2 (Biotest), detecting the MHC class II directed to IFN-7 (Fig. 2A). Treatment of cornea invariant or 7-chain; Tu 22 (Biotest) and Leu 10 fragments with neutralizing antibody alone had no (Becton-Dickinson), directed to HLA-DQ antigens. effect on MHC class II expression. The second and third step consisted of biotinylated To study the effect of IL-1 on the inductive effect of rabbit anti-mouse immunoglobulins and peroxidase- IFN-7,12 fragments of one cadaver cornea were incu- conjugated ABC, respectively (both purchased from bated in standard culture medium, in medium sup- Dakopatts, Copenhagen, Denmark). All incubations plemented with 100 U/ml IFN-7, or in medium con- were done for 30 min at room temperature and fol- taining a mixture of 100 U/ml IFN-7 and 100 U/ml lowed by a wash in three changes of phosphate-buff- IL-1 per ml medium. Incubation of corneal samples ered saline, pH 7.2, for 15 min. The reaction product with a mixture of IL-1 and IFN-7 did not alter the was developed by addition of 3-amino-9-ethylcarba- IFN-7-induced MHC class II antigen expression zole and H2O2. (Fig. 2B).

Results Discussion Parts taken from three cadaver corneas and from Using in situ immunohistochemical techniques one fresh cornea were placed in separate bottles con- and a panel of monoclonal antibodies, we showed taining 5 ml of culture medium to which IFN-7 had that IFN-7 is capable of inducing de novo expression been added in doses of 5000, 500, 100, or 50 U/ml. of MHC class II products on HCEC. Class II MHC Pieces from from other cadaver corneas were handled products13 can be divided into three major subsets of similarly, but they received IL-1/3 in doses of 50, 5, or products, called HLA-DR, HLA-DQ (or -DC), and 0.5 U/ml or IL-6 in doses of 300, 30, or 3 U/ml. HLA-DP (or -SB). The HLA-DR antigens were ini- Control HCEC, incubated in culture medium de- tially believed to occur only on cells involved in im- void of cytokines, did not react with any of the mono- mune responses, ie, B lymphocytes, activated T cells, clonal antibodies used (Fig. 1A). Similarly, neither monocytes/macrophages, dendritic cells, and vascu- IL-1 nor IL-6 induced MHC class II product expres- lar endothelial cells. In the last few years, however, an sion. Human IFN-7, on the other hand, induced de increasing number of nonlymphoid cell types has novo expression of MHC class II products and the been shown to display HLA-DR molecules either invariant or 7-chain of HLA-DR on HCEC in a constitutively14 or after appropriate stimulation by dose-dependent fashion. After treatment with 50 lymphokines.15 The HLA-DP and -DQ antigens, on U/ml, a mosaic-like, patchy staining was caused by the other hand, are more restricted in distribution16 scattered endothelial cells that expressed HLA-DR a- and are less easily induced by lymphokines. and jQ-chains (Fig. IB) and HLA-DQ chains on their In the normal human cornea, MHC class II prod- cell membranes and/or in the cytoplasm. With in- ucts are restricted to epithelial dendritic cells, occa- creasing doses of IFN-7, more endothelial cells ex- sional stromal spindle cells, and vascular endothelial pressed MHC class II products (Figs. 1C-1F), and cells in the limbus.1"3 However, in the course of after treatment with doses of 5000 U/ml, virtually all chronic infections4'7 or during allograft rejection,4-5 HCEC were found to be immunoreactive (Figs. "aberrant" HLA-DR expression may be found on the

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Fig. 1. Expression of MHC class II products in corneal endothelium upon induction by IFN-7. (A) Control endothelium, devoid of cytokines, lacks HLA-DR expression. (B) Scattered HLA-DR+ endothelial cells are observed after treatment with 50 U/ml IFN-7. (C, D) Upon treatment with 100 U/ml IFN-7, a mosaic, patchy immunoreactivity is observed for monoclonal antibodies LN2 (C)and TALI B5 (D). (E, F) Treatment with 500 U/ml IFN-7 reveals increased numbers of endothelial cells expressing HLA-DR antigens. {G, H) After incubation with 5000 U/ml IFN-7, virtually all corneal endothelial cells express the invariant or gamma-chain of HLA-DR (G) and HLA-DQ antigens (H). Three-step ABC-technique for TAL1B5 (A, B, D), CR3/43 (E), L243 (F), LN2 (C, G) and Leu 10 (H), slightly counterstained with Mayer's hematoxylin, all figuresorigina l magnification X40.

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2a 2b Fig. 2. Effect of neutralizing antibody (A) and 1L-1 (B) on the induction of MHC class II expression by IFN-7 in corneal endothelium. (A) Addition of an excess of neutralizing monoclonal antibody D9D10 to the medium containing 100 U/ml IFN-7 completely inhibits HLA-DR expression. (B) Addition of 100 U/ml IL-1 to the medium containing 100 U/ml IFN-7 does not alter MHC class II expression. Three-step ABC-technique for L243, counterstained with Mayer's hematoxylin, all figures original magnification X40.

cellular elements of the cornea. This expression is kines induces MHC class II expression in HCEC, al- thought to be due to induction by lymphokines, liber- though IL-6 did enhance MHC class I expression in ated by nearby T lymphocytes. these cells (data not shown), thereby confirming the In a previous, preliminary report17 we showed the results of a previous study.25 induction of HLA-DR antigens on human corneal The induction of MHC class II products by IFN-7 epithelial and endothelial cells by recombinant was specific since the addition of a neutralizing anti- IFN-7 in cryostat sections. The use of flat mounts of body completely abolished this activity of IFN-7. On sheets of HCEC in the current study allowed us to the other hand, we could not confirm the previously detect marked heterogeneity in the response of these reported inhibitory effect of IL-1 on the inducing ef- cells to IFN-7. Low doses of IFN-7 induced only a fect of IFN-7.12 few cells to express MHC class II products, whereas Consistent with earlier reports,4"717'26 these results high doses of IFN-7 induced a response in many cells. show that HCEC should be added to a growing list of This dose dependency was probably due to a variable nonhematopoietic cells that may express MHC class density of IFN-7 receptors, displayed at the cell II antigens after appropriate stimulation. MHC class membrane of HCEC. II antigens are instrumental in the activation of allo- The IFN-7 not only triggered the expression of geneic T cells and are primarily responsible for ini- HLA-DR and HLA-DQ antigens on HCEC, but in tiating allograft rejection. Induction of MHC class II addition, it induced the synthesis and expression of antigens on otherwise "silent" HCEC would explain the invariant or 7-chain of HLA-DR molecules, as why this corneal cell layer represents a major target in demonstrated with monoclonal antibody LN-2. the rejection process of corneal allografts. Moreover, the number of LN-2+ HCEC generally ex- The MHC class II expression by HCEC might also ceeded that of cells showing HLA-DR a- and /3- play a role in the immune response of ocular tissue to chains. Similar, noncoordinate expression of class II exogenous infections or allergenic agents. The pres- MHC chains has been observed in mice after in vivo ence of HLA-DQ molecules, normally required by treatment with IFN-718 and in humans,19 which monocytes to exert their antigen-presenting func- raises the question whether the 7-chain of HLA-DR tion,27 would make HCEC able to serve as antigen- molecules exerts a function unrelated to class II ex- presenting cells. pression, eg, in the intracellular transport of various The MHC staining of the HCEC layer after IFN-7 molecules.20 treatment revealed a striking heterogeneity of the cell In addition to IFN-7, other soluble factors pro- population. Studies of the cytoskeletal organization duced during an immune response, have been found of these cells have likewise revealed a distinct hetero- to regulate MHC class II expression.21 Both IL-1 and geneity.28 The physiologic or pathologic significance IL-6 are cytokines with a broad spectrum of immu- of this heterogeneity is unclear. Additional studies are nomodulating and inflammation-associated activi- required to determine whether it, in response to ties, including costimulation of T- and B-cell prolifer- IFN-7, has a relationship to heterogeneity in subcel- ation and induction of acute-phase proteins and lular structure. If so, it may be interesting to consider fever.22"24 We demonstrated that neither of the cyto- the possibility that the presence of nonresponsive

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cells is related to donor age. Alternatively, nonre- culture technique for growing corneal epithelial, stromal, and sponsive cells may be stochastically distributed be- endothelial tissues separately. Am J Ophthalmol 46:294, 1958. 12. Johnson WJ, Kelley A, Connor JR, Dalton BJ, and Meunier tween responsive cells and may (or may not) become PC: Inhibition of IFN-7-induced la antigen expression on sy- responsive with longer incubation time. Studies of novial fibroblastsb y IL-1. J Immunol 143:1614, 1989. this type, although relevant to improving transplant 13. Radka SF, Charron DJ, and Brodsky FM: Review: Class II technology, are restricted by the limited availability molecules of the major histocompatibility complex considered of human corneas. as differentiation markers. Hum Immunol 16:390, 1986. 14. 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