Cytokine mRNA expression in tolerant heart allografts after immunosuppression with cyclosporine, sirolimus or brequinar

Transplant Immunology 1997; 5: 189-198

Cytokine mRNA expression in tolerant heart allografts after immunosuppression with cyclosporine, sirolimus or brequinar Ling Tian, Stanislaw M Stepkowski, Xiumei Qu, Mou-Er Wang, Min Wang, Jiang Yu and Barry D Kahan Division of Immunology and Organ Transplantation, Department of Surgery, The University of Taas Medical School at Houston, Houston, Tms Received 24 April 1997; accepted 15 May 1997

Ahstractt We sought to examine the impact of the preferential activation of Th2 cells on the induction and maintenance of a tolerant state in heart allograft rat recipients treated with a short course of cyclosporine (C&4),sirolimus (SFU) or brequinar (BQR). A quantitative polymerase chain reaction (F’CR) method was used to measure the levels of cytokine mRNAs, namely interferon (IFN)-y and mterleukin (IL)-2 in T helper 1 (Thl) cells and IL-4, IL-5 and IL-10 in Th2 cells. Our main findings were that on day 5 postgrafting allografts from untreated recipients had increased levels of IFN-~(216 2 119 fg), IL-2 (449 f 75 fg), IL-4 (6.2 It 1.3 fg), IL-5 (34.8 k 9.3 fg) and IL-10 (1554 f 184 fg) mRNAs compared with normal hearts. CsA reduced the levels of IFN-x IL-2, IL-5 and IL-lo, but not IL-4, mRNAs. SRL did not affect the expression of cytokine mRNAs. BQR decreased the levels of IFN-x IL-2 and IL-lo, but not IL-5 or IL-4 mRNAs. Compared with grafts from untreated recipients, those from CaA- or BQR-treated tolerant hosts (day 100) displayed undetectable IL-2 mRNA levels, and reduced levels of EN-): IL-4 and IL-10 mRNAs. In fact, the patterns of cytokine mRNA expression in grafts from CSA- and BQR-treated tolerant hosts were similar to those of normal hearts. Grafts from SRL-treated tolerant hosts merely showed slightly increased Th2 cell activity. In conclusion the selective activation of Th2 cells is not absolutely required for induction or maintenance of tolerance.

Introduction T helper 1 (Thl) cells have been characterized by their production of interleukin (IL)-2, interferon-y (EN-y) antitumour necrosis factor (TNF), whereas Th2 cells have been characterized by their production of IL-4, IL-S, IL-6, IL-10 and IL-13.rs2 The activation of Thl cells promotes the generation of T delayed-type hypersensitivity (DTH) cells and T otoxic (Zz) effector cells, which mediate allograft destruction. cytIn contrast, the activation of ‘Ih2 cells stimulates the preferential production of IL-4 and IL-lo, to which the induction and maintenance of transplantation tolerance have been attributed.4 Heart allografts from mice rendered tolerant by pretreatment with donor-specific blood transfusion, or by pet-i-operative treatment Address for correspondence: BD Kahan, Division of Immunology and Organ Tkansplantation, Department of Surgery, The University of Texas Medical School at Houston, 6431 Fannin, Suite 6.240, Houston, TX 77030, USA. 0 Arnold 1997

with anti-CD4 monoclonal antibodies (MAb), or cyclosporine (CsA) showed a greater than 90% reduction in the expression of IFN-yand IL-2 mRNAs compared with those from untreated recipients.’ The same tolerant allografts expressed IL-4 and IL-10 mRNAs levels similar to those of untreated grafts, suggesting preferential activation of Th2 cells4 These findings were recently confumed by studies in which mice were rendered tolerant to allogenic pancreatic islets by anti-CD4 MAb, and rats were rendered tolerant to heart allografts by a short course of anti-CD4 MAb or sirolimus (SRL).s-lo However, allogenic pancreatic islets from transgenic mice that overexpress IL-10 were promptly rejected in spite of the increased local production of IL-10 by transplanted pancreatic islets.” Moreover, treatment with a long-acting form of recombinant IL-10 failed to delay the rejection of pancreatic islet allografts.12 Furthermore, transgenic mice that overexpress IL-4 displayed an increased production of EN-y, which was strongly associated with cell-mediated allograft rejection.13 In fact, IL2 knockout mice that continuously produce IL-4 in the absence 0%6-3274(97)TI181OA

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of IL-Z failed to develop tolerance to allograft~.‘~ Thus, although the Thl/Th2 paradigm may be useful to discern cytokine regulatory patterns during rejection and the induction of unresponsiveness processes, it is unclear whether preferential activation of Th2 cells is mandatory for induction of transplantation tolerance. Donor-specific transplantation tolerance is readily induced in rats by a short course of CsA, SRL or brequinar (BQR), in spite of the fact that these agents have completely different mechanisms of action.‘0*‘““6 CsA, a hydrophobic cyclic endecapeptide, blocks the T cell production of IL-2 and other cytokines.17-‘g SRL, a hydrophobic 31-membered macrocyclic lactone, inhibits T cell responses by blocking IL-2, IL-4 and IL6 cytokine signal transduction.‘sZ BQR, 6-fluoro-2-(2’-1-1’biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid, inhibits de novo pyrimidine biosynthesis by interfering with the activity of dihydro-erotic acid dehydrogenase.% We used quantitative reverse-transcriptase polymerase chain reactions (PCR) to measure cytokine mRNA expression during the induction of transplantation tolerance. On day 5 postgrafting, heart allografts from recipients treated with CsA, SRL or BQR displayed different patterns of cytokine mRNA expression. However, heart allografts from tolerant recipients (day 100 postgrafting) treated with CsA or BQR had nearly identical patterns of cytokine mRNA expression as those from normal control animals. Interestingly, heart allografts from SRL-treated tolerant recipients showed only slightly increased Th2 cell activity compared to normal hearts. Thus, we found that no pathognomonic pattern of cytokine mRNA expression is associated with transplantation tolerance.

(10.0 mg/kg/day) was delivered iv by a 14-day osmotic pump. BQR was freshly dissolved in distilled water (4.0 mg/ml) and delivered at a dose of 8.0 mgikg/dose per oral gavage every second day from the day of transplantation until day 28. Heart transplantation BUF donor hearts were heterotopically transplanted into the abdomens of WF recipients by the modified method of Ono and Lindsey.= Graft function was evaluated daily by transabdominal palpation, and rejection was defined as cessation of the heart beat.

We sought to examine the impact of a short course of CsA, SRL or BQR on the preferential activation of Th2 cells during the induction and maintenance of transplantation tolerance.

Total RNA ad cDNA preparation Total RNA was extracted from the heart tissue by the guanidine isothiocyanate caesium chloride method.% Hearts were diced coarsely, rinsed in ice-cold saline and immediately frozen in liquid N2. Tissues were homogenized in guanidine isothiocyanate solution [4 M guanidine isothiocyanate, 25 mM sodium acetate (pH 6), 0.8%, pmercaptoethanol]. Samples were layered on a 5.7 M caesium chloride gradient and ultracentrifuged for 21 h at 32 000 rev/min. After precipitation, the RNA was washed with 70% ethanol and measured spectrophotometrically at 260 and 280 nm; the 260/280 ratios consistently ranged from 1.7 to 2.0 for all examined samples. The cDNA was synthesized in a reverse transcription reaction as previously described.” Briefly, 2 pg total RNA was incubated for 5 min at 65°C with 0.5 pg oligo (dT)ls. chilled on ice for 3 min, then incubated for 60 min at 42°C with 1 U RNasin, 1 mM dNTPs @omega, Madison, WI, USA) and 10 U reverse transcriptase (Perk&Elmer, Branbhburg, NJ, USA), in a 20 l.d reaction. The reaction tube was heated to 99°C for 5 min and quickly chilled on ice. The cDNA was diluted to a final volume of 100 pl and stored at -70°C as the stock for all PCR reactions. The oligo (dT)15 primer was used for first-strand cDNA synthesis (Fisher, Pittsburgh, PA, USA). The RNA samples did not contain genomic DNA encoding cytokines, because the PCR with specific primers for cytokines and RNA samples produced no specific bands.

Materials, methods and experimental design

Construction of competitive DNA hagments for measurements of cytokines Six competitive standard DNAs (sDNA) were constructed

Objective

Animals

Wistar Furth (WF; RTl”), Buffalo (BUF; RTlb) and Brown Norway (BN, RTl”) male rats (7-9 weeks old) were obtained from Harlan Sprague-Dawley (Indianapolis, IN, USA). Rats were housed in wire-bottomed cages with controlled light/dark cycles, and allowed free access to water and rat chow in the Animal Care Center of the University of T&as Medical School at Houston.

SRL (Rapamune@; 19-66 mg/ml) was obtained from WyethAyerst (Rouses Point, NY, USA) and diluted with a vehicle of 10% Neen 80, 20% N,N-dimethylacetamide and 70% polyethylene glycol400. After the 14-day 2002 osmotic pump (Alzet, Palo Alto, CA, USA) was primed in a 0.9% NaCYO.01 M phosphate buffer solution at 37”C, the SRL (0.8 mg/kdday) was loaded into the chamber and infused intravenously (iv) via the recipient’s femoral vein. CsA was obtained in powdered form (SandimmuneTM; Sandoz, Basel, Switzerland) for dissolution in a mixture of 1% alcohol and 99% cremophor. The CsA l?ansplant Immunology 1997; 5: 189-198

to contain sense and antisense primer sequences specific for rat, pactin, IFN-x IL-2, IL-Q, IL-5 and IL-10?7-31 A retrovirus GlO gene served as a template for the PCR reaction in which two composite primers were used. Each composite primer contained the cytokine’s gene primer sequence attached to a short stretch of nucleotides that hybridized to opposite strands of the GlO gene DNA fragment. PCR products were separated on agarose gels and subcloned in TA cloning vectors (Invitrogen, San Diego, CA, USA). Plasmids containing each competitive sDNA insert were purified. Concentrations of sDNAs were measured on a spectrophotometer to prepare the lo-fold stock dilutions ranging from 1000 to 10 fg (femtogram: 1O-15gram). Measurement of mRNAs by competitive F’CR

Oligonucleotide primers for pactin, IFN-x IL-2, IL-4, IL-5 and IL-10 were designed based on genomic sequences. The primers included: Bactin, 5’ ATG GAT GAC GAT ATC GCI’G 3’ and 5’ ATG AGG TAG TCT GTC AGGT 3’; IFN-K 5’ ATC TGG AGG AAC TGG CA4 AAG GACG 3’ and 5’ CCT TAG GCT AGA ‘I-I-C TGG TGA CAGC 3’; IL-2,SAAC AGC GCA CCC ACT’ TCAA 3’ and 5’ CAG ATG GCT ATC CAT Cl-CC 3’; IL4,5’ AAC ACC ACG GAG AAC GAG Cl-C ATC 3’ and 5

Cytokine mRNA expression in tolerant heart ailografts after immunosuppression

AGT GAG TTC AGA CCG CTG ACA CCT 3’; IL-S, 5’ CCT TGC TGT ACT GTC cTGA3’ and 5’ CAG GGT CTC GAT CIT AGcT3’; and IL-lo, 5’ CAT GCC TGG C-K AGC ACT 3’ and 5’ GGG AAC TGA GGT ATC AGA 3’. A constant concentration of experimental cDNA (2.5 ~1) was co-amplified with serial lo-fold dilutions of known concentrations of sDNA using appropriate antisense and sense primer pairs in a 50 pJ reaction mixture to obtain an estimate of the concentration of experimental cDNA present in each sample. A second two-fold titration over a narrower range of sDNA concentrations was performed for precise quantitation. The amplification reactions, which were performed in a 9600 DNA thermal cycler (Perkin-Elmer), included 35 cycles of denaturation at 94°C for 15 s, annealing at 60°C for 1 min and extension at 72°C for 45 s. The PCR mixture included 0.125 lg each of sense and antisense primers, 160 pM dNTPs, 1.5-2.0 pM magnesium chloride, 1.25 U Taq DNA polymerase and 1 x PCR buffer, as recommended by the manufacturer (Perkin-Elmer) in a reaction volume of 50 l.d. The products of amplification (20 fl) along with molecular weight markers were analysed by 2% agarose gel electrophoresis at 120 V after adequate separation of the experimental cDNA and sDNA products. The bands were visualized by ethidium bromide staining and photographed under UV light. Polaroid photographs (Polaroid, Cambridge, MA, USA) and scanned gel images were plotted on a computer with the Image program (NIH, Washington, DC, USA). The results are presented in femtograms as mean f SD from three or four independent experiments on different animals. Limiting dilution analysis

Xvelve replicates of two-fold serial dilution of responder cells (ranging from 400 to 25 600 cells) with 5 x lo4 irradiated (2000 rads) BUF splenic stimulator were cultured for seven days in complete-RPM1 1640 medium (Sigma Chemical Co., St Louis, MO, USA), which was supplemented with 10% heat-inactivated foetal calf serum (FCS) and 10 unit/ml of purified IL-2 (Collaborative Research Inc., Bedford, MA, USA). The cytotoxic activity of each well was assessed by a 5-h incubation with

,

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cDNAlstutdwd 557 I 480

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1 x lo4 “Cr (Na&‘1Cr]04, specific activity 200-500 mCi/mg Cr; Amersham, Arlington, IL, USA)-labelled BUF Morris hepatoma 7316 target cells. “Cr release in the supematant fluids was measured using a gamma counter. Values that exceeded the mean spontaneous chromium release by more than three SD were considered positive. Minimum cl&square estimates of the frequency ofalloantigen-specific Tc cells (m) with 95% confidence limits were calculated by the relationship between responder cell number and the natural logarithm of the fraction of negative cells.

Results Quantitative competitive PCR method The sDNAs were constructed to show approximately

a lOO-bp size difference from experimental cytokine cDNA for pactin, IL-2, IL-4, IL-10 and IFN-yand approximately a 40-bp difference for IL-5 (Figure 1). The different sizes of the cDNA and sDNA products were distinguished by agarose gel electrophoresis using densitometly measurements (Figure 2A, B). The point of equivalence where the density ratio of experimental DNA/sDNA = one defines the amount of experimental DNA present in the original samples.

In ufuo transplant models Untreated WF recipients rejected BUF heart allografts at a mean survival time (MST) of 6.5 1 0.5 days. A 1Cday course of treatment with daily SRL doses (0.8 mg/kg/day) delivered iv by continuous infusion prolonged heart allograft survival to 75 f 18.9 days with six out of 18 grafts surviving for more than 100 days. Recipients treated for 14 days with CsA (10 mg/kg/day) delivered by osmotic pumps displayed an MST of 65.5 +- 20.8 days with five out of 10 grafts surviving for more than 100 days. Similarly, recipients treated every second day (qod) for 28 days with BQR (8 mg/k@dose) had an MST of 82.5 r 31 days with six out of 10 heart allografts functioning for more than 100 days.

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Fllre 1 The comparison of the difference in size between the polymerase chain reaction (PCR) products amplified from native complementary DNA (cDNA) and artificial standard DNA @DNA) for Factin, IL-2, IL-4, IL-S, IL-10 and IFN-y The first lane shows a 1 kb marker (M) and the other lanes show cDNA (C) versus sDNA (S) for pactin, IL-2, IL-4, IL-5, IL-IO and EN-y. For experimental details see the Materials and methods section.

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B.

Figure 2 Densitometry measurements of IL-10 mRNA expression. (A) The polymerase chain reaction (FCR) products, stained by ethidium bromide on agarose gel, represent IL-10 standard DNA (sDNA) quantities of 500,250,125 or 62.5 fg, in comparison to a constart quantity of experimental complementary DNA (cDNA). (B) A two-fold titration of sDNA and a constant amount of experimental cDNA are analysed by densitometry and presented as the plot of the sDNA/cDNA ratio versus sDNA concentrations (500,250,125 and 62.5 fg).

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FIgnre 3 Expression of IFN-): IL-2, IL-4, IL-5 and IL-10 mRNAs in normal Buffalo (BUF; RTlb) hearts, syngenic Wistar Forth (WF; ml”), and BUF heart allografts from untreated WF recipients. The results are presented as a mean 2 SD (three to five experimental animals per group). For experimental details see the Materials and methods section.

Transphnt Immumlogy 1997; 5: 189-198

Cytokine mRNA expression in tolerant heart albgmfts

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Flgurr 4 Expression of IFN-x IL-Z, IL-4, IL-5 and IL-10 mRNAs in heart allografts from sirolimus (SRL), cyclosporine (CM) or brequinar (BQR) treated recipients on days $10 and 20 after transplantation. Details as in Figure 3.

Transplant Immunology 1997; 5: 189-198

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Figure 5 w of EN-Y; IL-5 IL4, IL-5 and IL-10 mRNAs in heart allografts from sirolimus (SRL), cyclosporine (CSA) or brequinar (BQR) treated recipients on day 60 or 100 after transplantation. Details as in Figure 3.

The animals with well-functioning (> 100 days) heart aliografts subsequently accepted donor-type BUR but not third-party BN, heart alIografts. Heart ahografts from these immunosuppressed recipients were used to quantify the expression of cytokine mRNAs on days 5, 10, 20, 60 and 100 ~st~a~g_ These values were compared to cytokine mRNA levels in normal nontransplanted BUF hearts, or in syngenic WF and ahogenie BUJ? heart grafts transplanted into untreated WF recipients (five days postgrafting). Transplant Immunology 1997; 5: 189-198

Expression of &a&in, IFNq, IL2,1L4, Il.4 and Ii.40 mRMs duningor bmediateiy after lreatment arith SRL, ChA or BQR No signiticant difference was observed in the concentrations of pactin mRNA among all hearts. Normal BUF hearts had low levels of IFWy(5.3 + 0.3 fg), IL-4 (2.7 z!z0.5 fg), IL-S (12.0 & 5.5 fg) and IL-10 (36.1 1(37.9 fg; Figure 3) mRNAs, but an undetectable level of IL-2 mRNA. Compared with normal BUF hearts, at day 5 postgrafting, syngenic WF grafts showed

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Days Figure 6 Kinetics of cytokine mRNA expression during induction and maintenance of transplantation tolerance. Tbe dashed line (- - -) represents a minbnum cytokine mRNA expression when measured in normal hearts, and the dotted line (s-e) represents a maximum cytokine mRNA expression when measured in untreated controls on day 5 postgraftmg @); both lines are compared with cytokine mRNA expression in heart atlografts from recipients treated with sirolimus (SRL; +), cyciosporine (Cc& 0), or brequinar (BQR; A). Detaiis as in Figure 3. slightly increased concentrations

of only IL-10 mRNA (335.0 + 75.7 fg,p < 0.05; Figure 3). In contrast, compared to normal BUF hearts, BUF heart ahografts in untreated WF recipients on day 5 postgrafting expressed increased levels of IFN-~(216.0 2 119 fg;p < 0.03), IL-2 (449.0 f 75 fg;p e O.OOOl),IL-4 (6.2 + 1.3 fg;p < O.Ol), IL-S (34.8 + 9.3 fg;p c 0.014) and IL-10 (1554.0 & 184 fg;p c 0.0001) mRNAs (Figure 3). Compared with BUF ahografts from untreated recipients, on day 5 postgrafting, BUF heart ahografts from recipients treated with SRL had a similar pattern of expression of IFN-~(331.0 f 80 fg;p c O-01), IL-2 (478.0 f 132 fg; NS), IL-4 (4.0 f 0.9 fg; NS), IL-5 (13.2 ,+ 4.6 fg; NS) and IL-10 (1956.0 & 579 fg; NS) mRNAs (Figure 4). In contrast, CsA therapy reduced the expression of IIW~(31.6 f 3.2 fg;p c O.OOl), IL-2 (88 f 44 fg;p < O.OOl), IL-5 (6.6 f 2.8 fg;p < 0.02) and IL-10 (402.0 + 109 fg; p < 0.04) mRNAs, but not the expression of IL-4 (4.4 + 2.0 fg; NS) mRNAs. Compared with hearts from untreatTransplant

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ed recipients, those from recipients treated with BQR showed a reduced level of IlWy(80.0 rf: 41 fg;p < O-01), IL-2 (240.0 + 27 fg;p < 0.01) and IL-10 (269.0 + 42 fg;p < 0.02) mRNA expression; BQR did not appear to affect the expression of IL4 (4.2 + 1.1 fg; NS) or IL-5 (50.8 + 12 fg; NS) mRNAs. Ten days after postgrafting there continued to be some rather small differences in cytokine mRNA profiles among grafts from hosts treated with different immunosuppressants. In particular, the SRL group had higher IL-10 mRNA levels (2498 f 1533 fg; Figure 4) than both the CsA and the BQR groups, and the BQR group had higher IL-5 mRNA levels (104 + 35.1 fg) than both the SRL and the CSA groups. However, 20 days after postgrafting heart ahografts from animals treated with SRL, CsA or BQR had similar levels of EN-y, R-2, IL-4, IL-5 and IL-10 mRNAs (Figure 4). Thus, CsA and BQR, but not SRL, affected Thl- and ThZ-mediated cytokine mRNA expression shortly (day 5) after grafting.

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Ekpreaion of IFN-y, IL-Z, IL4 and IL10 mRNAe in lon*tmm amruidng cecipientta treated with SRL, CsA or BQR The expression of cytokine mRNAs was also assessed at 60 and 100 days postgrafting in extracts of well-functioning BUF heart allografts from hosts treated with SRL, CsA or BQR (Figure 5). On one hand, the tolerant hearts in all three treatment groups had signitlcantly reduced levels of IFN-yand XL-2 mRNAs compared with those of the untreated recipients, On the other hand, the cytokine mRNA levels in the tolerant hearts were similar to those observed in normal nongrafted controls. In particular, on day 100 postgrafting, grafts in SRL treated recipients had a mean IFN-ymRNA concentration of only 4.6 4 1.1 fg, and an undetectable IL-2 mRNA level; in CsA-treated group, the mean IFN-ymR.NA level was 4.0 z!z1.0 fg and the IL-2 mRNA level was undetectable; and in BQR-treated group, the mean IFN-y mRNA level was 3.5 2 1.0 fg, and the IL-2 mRNA level was undetectable (Figure 5).

Gn day 100, the Th2 cells proved only modestly active in heart allografts from tolerant hosts. Grafts from the U&-treated group showed a mean IL-4 mRNA level of 7.8 4 0.6 fg, an IL5 mRNA level of 36.9 2 4.6 fg, and an IL-10 mRNA 1eveI of 99 -t 38 fg. Grafts from the C&-treated group had mean IL-4, IL5 and IL-10 mRNA levels of 2.8 f 0.5,43 k 7.1 and 81.2 + 15.5 fg, respectively. Finally, grafts from the BQR-treated group had mean IL-Q, IL-5 and IL-10 mRNA levels of 2.6 + 0.3,28.4 & 2.5 and 27.3 2 9.3 fg, respectively (Figure 5). Thus, the cytokine mRNA profiles of Th2 cells in tolerant BXJF grafts from the CsA- or BQR-treated groups were almost identical as those detected in normal BUF hearts, nameiy iow levels of IL-4 and IL-10 mRNAs, and slightly elevated levels of IL-5 mRNA @ c 0.03; Figure 6). Furthermore, compared to normal hearts, tolerant grafts from the SRL-treated group had only slightly higher IL-4 @ < O.OOl), IL-5 (p < 0.02) and IL-10 mRNA @ c 0.05) ~n~ntrations (Figure 6).

Figure 7 Correlation between immunological status and the frequenw of alloantigen specific T cytotoxic cells (flk). The fTkwas examined in lymph nodes fkom normal rats {O),from recipients which remained untreated (A), or from recipients which were treated with si&mus (SW 0), cyclosponhe (CSA; l), or brequinar (BQR l)* The flE was examined on day 5 ~t~~~g in response to donor-type Bnffalo (BUF; Wlb) (A) or third-party Brown Norway (BN, FtTln) (B) alloantigens, as we11as in response to donor-type BtJF alloantigens on day 60 postgrafting (C). For experimental details see the Materials and methods section.

Tmnspkmt Immunology 1997; 5: 189-198

Cytokine mRNA expression in tolerant heart allografts after immunosuppresion Fmquency of allomtisen-speciflc Tc cells in SRL, CSA- end BQR-treated recI~ents The immunological status of recipients was tested by analysis of the fTc. Normal WF lymph node cells displayed an fib of antiBUF-directed elements of 1:4718 fi 1327 (Figure 7). On day 5 postgrafting, grafts from untreated recipients showed a sevenfold increase in the fR (1:619 +. 85). Compared to the fR in normal lymph nodes, the fTc values in the SRL (1:1987 +- 852) and CsA (1:1116 + 338) treated groups were slightly increased, whereas the flh value was similar in the lymph node cells of hosts treated with BQR (1:5395 + 2068). On day 60 postgrafting, compared to normal hearts, tolerant recipients showed either reduced (SRL group, 1:8997 + 1744) or similar (CsA group, 1:5240 + 999; BQR group, 1:7030 + 1020) fR values. These results suggest that compared to untreated controls, tolerant recipients had a similarly reduced immune status.

Discussion The quantitative PCR method was used to measure the expression of several cytokine mRNAs at the graft site during the induction of transplantation tolerance after the administration to rats of a short course of CsA, SRL or BQR. On day 5 postgrafting allografts from untreated recipients had increased concentrations of EN-y, IL-2, IL-4, IL-5 and IL-10 mRNAs compared with syngenic grafts or native hearts. Although SRL monotherapy protected grafts from rejection, on day 5 postgrafting cytokine mRNA expression was similar in the grafts of SRL-treated recipients compared with tissues in hosts experiencing allograft rejection. In contrast, on day 5 postgrafting, CsA monotherapy inhibited the expression of IFN-x IL-2, IL5 and IL-10 mRNAs by four- to seven-fold, but it did not affect IL-4 mRNA concentrations. Furthermore, BQR monotherapy reduced the expression of IFN-yand IL-2 mRNAs by two-fold and IL-10 by six-fold, did not affect IL 4, but increased IL-5 mRNA expression by 1.5-fold. Thus, drugs that act by distinct molecular mechanisms produced different effects on cytokine mRNA expression during the early induction phase of tolerance. These unique patterns may reflect each drug’s effects on transcription, translation and post-translational modification of individual cytokine genes. These findings confirm and extend the results of a previous study in which a nonquantitative PCR method was used to fmd that SRL did not inhibit the expression of IFN-x IL-2 or IL-10 mRNAs in heart allografts in nonsensitized recipients.‘0 In contrast, in presensitized hosts challenged with donor-type heart allografts, SRL mitigated the strong expression of intragraft R-2, but not IL-4 or IL-10 mRNAs between 3 and 6 h postgrafting. In these sensitized recipients, a seven-day course of SRL prolonged graft survivals to 50 dayss2 Thus, therapeutic doses of SRL do not inhibit Th2 cell activity in vivo in either unsensitized or sensitized rat recipients32 or in vitro in mouse lymphocytes.33 In contradistinction, it has been demonstrated by Northern blot analysis, and nonquantitative as well as quantitative PCR and in situ hybridization methods that CsA reduces the concentrations of IFN-)I and IL-2 mRNAs after allogenic challenge.3”6 Similarly, lymphocytes from CsA-treated human patients bearing stable kidney transplants show decreased levels of IFN-r and IL-2 mRNAs.35 Thus, the quantitative results presented herein confirm previous studies that therapeutic CsA doses significantly reduce but do not completely block the expression of cytokine mRNAs. Transplant Immunology 1997; 5: 189-198

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However, much less work has been reported about the effects of BQR on the expression of cytokine mRNAs. It is well documented that a short course of BQR induces permanent acceptance of heart, kidney, liver, or small bowel allografts in rats3’ A semi-quantitative PCR method was used to demonstrate that this tolerant state correlated with reduced graft site expression of IL-2, IL-4, IL-6, IL-10 and TNF-a mRNAs. The present study con6nned the inhibitory effect of BQR on IFN-x IL-2 and IL-10 mRNA expression, and showed a selective increase of IL5 mRNA expression. The latter observation suggests that BQR may potentiate the production of IL-5. Short-term immunosuppression with CsA, SRL or BQR induced donor-specific transplantation tolerance. On day 100, all tolerant grafts had undetectable IL-2 and low IFN-y (2.5-5.0 fg) mRNA levels, like normal hearts. Furthermore, the concentrations of IL-4 mRNA in tolerant hearts were similar to those in normal hearts, and just a little lower than those in grafts from untreated recipients. Overall IL-4 mRNA levels ranged in all examined animals between only 2.5-8.0 fg, suggesting small differences in the number of IL-4 mRNA copies observed in normal hearts and allografts undergoing rejection. However, compared to grafts from untreated recipients, the levels of IL-10 mRNA in tolerant grafts were significantly reduced by as much as 16- to 51-fold to 30-100 fg. In fact, the levels of IL-10 mRNAs in tolerant grafts were almost identical to those present in normal heart tissues. Only grafts from SRL-treated tolerant recipients showed slightly increased Th2 cell activity compared with normal hearts. Thus, we observed no evidence of an association between preferential activation of Th2 cells and transplantation tolerance. In contrast, many recent publications have suggested that the maintenance phase of tolerance correlates with the dominant activation of Th2 cells as well as with a reduced immune st.atu~.‘~~ For example, in CM-treated recipients transplantation tolerance was associated with a reduction in alloantigen-specillc fR and the development of T regulatory cells, which adoptively transferred the tolerant state.” In contrast, the SRL-induced tolerant state was transferred to virgin hosts with serum or its IgG fraction but not with T cell~.‘~ Heart allografts from these SRL-treated tolerant hosts showed undetectable IL-2 mRNA levels in Thl cells and active Th2 cells, as suggested by the presence of IL-4 and IL10 mRNAs (measured by nonquantitative PCR method).” However, we documented that although the alloantigen-specific fl’k cells observed in lymph nodes of CsA-, SRL- or BQR-treated tolerant recipients were similar to those in normal animals, the tolerant status did not correlate with a preferential activation of Th2 cells, as was shown by the quantitative PCR method. In fact, we observed no consistent pattern in Thl/Th2 cell activities that may be attriiuted to the early induction or late maintenance phases of transplantation tolerance. Acknowledgement This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK 38016-10).

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