Interleukin-15 gene transcripts are present in rejecting islet allografts

INTERLEUKINS

ELSEVIER

Interleukin-15 Gene Transcripts Islet Allografts R.C. Manfro, P. Roy-Chaudhury, and T.B. Strom

Are Present in Rejecting

X.X. Zheng, J. Steiger, P.W. Nickerson,

I

Y. Li, W. Maslinski,

L-2-DEFICIENT mice reject islet allografts in a T-celldependent fashion,’ proving that factors other than IL-2 can participate in rejection. IL-15, a macrophage product, is a recently described cytokine that supports T-cell growth in vitro and whose effects on T-cell proliferation are not blocked by cyclosporine.2.” The IL-15 receptor is a trimolecular complex consisting of a unique alpha chain as well as the beta and gamma chains of the IL-2 receptor.4 Common utilization of the IL-2 receptor beta and gamma chains is responsible for the similar biologic activities of IL-2 and IL-15.4 We hypothesized that IL-15 might participate in T-cell proliferation and rejection in an IL-Zdeficient system. To test this hypothesis we have performed islet allografts in IL-2 (-/-) knock-out (IL-2 KO) and IL-2 (+/+) wild type (IL-2 WT) mice and analyzed the expression of mRNA encoding for IL-15 using quantitative RTPCR.

Five micrograms of graft RNA was reverse transcribed into cDNA. For the PCR, 1125 of the cDNA product was combined with gene-specific murine IL-15 oligonucleotide sense (S’GACAC CACTITATCAACTG3’) and antisense (5’TGGACAATGCG TATAAAG3’) primers and Tuq DNA polymerase. Samples were normalized for the presence of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as described.6 To semiquantitative the relative amounts of gene transcripts between various samples, the individual PCRs were performed by coamplifying the cDNA of interest (wild type; WT) with an internal PCR control (a genespecific competitive template (CT) cDNA) containing a 47 basepair deletion (bases 36 to 83 bases of the cDNA encoding murine IL-15. By coamplifying the graft cDNA with a constant amount of the gene-specific competitive template, we determined the relative amounts of graft cDNA in the various samples as described.6 The amount of cDNA of interest is presented as the ratio of WT cDNA to CT.” A nonparametric analysis of variance method (KruskalWallis) was used. A significance level of P < .05 was required for statistical significance.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Six- to S-week-old DBA/ZJ mice (H-2d) were used as islet donors for IL-2 KO and WT C57Bl6xOla 129 (H-2b) background mice. IL-2 knock-out animals were created by the insertion in reverse orientation of the neomycin resistance gene in the third exon of the IL-2 gene.’ Recipient mice were rendered diabetic with a single intraperitoneal injection of streptozotocin (225 mg/kg). As a con trol, syngeneic islets obtained from C57Bl6xOla 129 IL-2 WT mice were transplanted in both KO and WT animals. Primary graft function was defined as a blood glucose under 200 mg/dL on day 3 posttransplantation, and rejection was defined as previously reported.’ RNA was isolated from snap-frozen graft tissue obtained from recipients sacrificed at the time of maximal T-cell infiltration. which corresponds to day 8 in WT animals and day 12 in KO animals.’

Heightened IL-15 gene expression is present in the allografts of IL-K0 (ratio WT:CT = 0.42 ? 0.03) and IL-2 WT (ratio WT:CT = 0.33 ? 0.04) as compared to syngeneic transplants (ratio WT:CT = 0.24 i- 0.03); (P < .Ol;

0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

From the Department of Medicine, Division of immunology, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts. Supported by NIH. Address reprint requests to Terry B. Strom, Department of Medicine, Beth Israel Hospital RE 319, Harvard Medical School, 330 Brookline Ave, Boston, MA 02215.

0041-1345/97/$17.00 PII SO041 -1345(96)0041 O-l

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Transplantation

Proceedings,

29, 1077-l 078 (1997)

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allogeneic either type versus syngeneic). Furthermore, IL-2 KO grafts bear a significantly higher amount of IL-15 mRNA than grafts from IL-2 WT mice (P < .0.5; IL-2 KO versus IL-2 WT cDNA:CT ratios). Next we tested whether splenic mononuclear leukocytes (MNLs) isolated from WT and KO animals respond to IL-15 after activation with anti-CD3 mAb. Indeed, antiCD3-activated MNLs from both WT and KO animals proliferate in response to IL-15 MNLs from the IL-2 KO mice do not proliferate in response to anti-CD3 stimulation alone due to lack of endogenously produced IL-2. Additive effects of other T-cell growth factors including IL-4 and IL-7 were also observed in these proliferation assays. In short, IL-15 gene transcripts are upregulated in islet transplant rejection in both WT and IL-2 KO recipients. Clearly T-cell growth factors other than IL-2, such as IL-4, IL-7, and IL-15 are able to maintain T-cell proliferation and differentiation and mediate allograft rejection in vivo.’ We

MANFRO,

ROY-CHAUDHURY,

ZHENG ET AL

postulate that IL-15 may cause or participate in IL-2independent T-cell activation. Finally, additive effects of IL-15, IL-4, and IL-7 on T-cell proliferation indicate that efficient therapeutic intervention into T-cell-mediated graft rejection may require targeting or common pathways of cytokine-triggered signal transduction.

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