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Experimental Biology and Medicine 228:466-471 (2003)
© 2003 Society for Experimental Biology and Medicine

HEME OXYGENASE

Generation of Heme Oxygenase-1-Transgenic Rats

C. Braudeau*, D. Bouchet*, C. Toquet{dagger}, L. Tesson*, S. Ménoret*, S. Iyer{ddagger}, C. Laboisse{dagger}, D. Willis§, A. Jarry{dagger}, R. Buelow{ddagger}, I. Anegon1,2,* and C. Chauveau1,*

* Institut National de la Santé Et de la Recherche Médicale (INSERM) U437/Institut de Transplantation Et de Recherche en Transplantation (ITERT) and
{dagger} Service d’Anatomie Pathologique, 44093 Nantes Cedex 01, France;
§ University College of London, London WC1E 6BT, UK; and
{ddagger} SangStat Corporation, Fremont, California 94555

Abstract

Heme oxygenase-1 (HO-1) expression protects cells from a variety of cellular insults and inhibits inflammation. However, its role in the regulation of immune responses has not yet been clearly established. We generated HO-1 transgenic rats to directly test the impact of HO-1 on the different immune mechanisms. To temporally control the expression of HO-1, we used a one-plasmid tetracycline (tet)-inducible system. This plasmid contains the H-2Kb promoter, which transcribes the tet transactivator (tTA) and expression of a human HO-1 cDNA is obtained in the absence of tetracycline. The DNA construct was microinjected into one-cell rat embryos and mothers and pups were maintained with tetracycline. Eight transgenic founders were obtained. Analysis of transgene expression in the absence of tet showed that 2 lines (12.4 and 12.6) expressed HO-1 mRNA in several organs (as detected by reverse transcription polymerase chain reaction) and at the protein level only in the thymus. Expression levels of transgene-derived HO-1 increased after withdrawal of tet compared with transgenic rats maintained with tet, as detected by analysis of mRNA levels by quantitative real-time reverse transcription polymerase chain reaction. Gross examination and histopathological analysis of several organs in both lines showed no anomalies. Thymocytes and splenocytes of both lines showed normal cell subpopulations and allogeneic proliferation compared with controls. Systemic immune responses against cognate antigens were normal in both lines, as evaluated by the proliferation of lymph node cells and the production of antibodies against keyhole limpet hemocyanin after immunization. Animals from line 12.6 rejected transplanted allogeneic hearts with the same kinetics as controls. In conclusion, short-term induction of HO-1 overexpression did not modify immune responses compared to those of control non-transgenic animals.

Key Words: HO-1 • transgenic rats • immune response • transplantation

Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the oxidative degradation of heme into biliverdin, free iron, and carbon monoxide. This 32-kDa stress-inducible protein provides protection against a variety of cellular injuries, such as oxidative stress, proinflammatory cytokines, and proapoptotic inducers (1–3). HO-1 has been shown to have anti-inflammatory actions (2), to suppress macrophage activation (4), to mediate the anti-inflammatory effects of interleukin-10 (5), and to inhibit allograft and xenograft rejection (2, 6, 7). However, whether HO-1 modulates antigen-specific immune responses or other lymphocyte functions has not yet been established. Previous publications describing transgenic mice for HO-1 have used promoters that are specific for neurons (8), vascular smooth muscle cells (9), lung cells (10), or cardiomyocytes (11, 12).

The aim of this study was to generate HO-1-transgenic rats to directly analyze the impact of HO-1 on various immune functions. We used the ubiquitous H-2Kb promoter to target HO-1 expression primarily to endothelial cells and leukocytes (13) and the tet-off inducible system (expression in the absence of tet) to control transgene expression. We obtained two lines of transgenic rats that, after 10 days of tet withdrawal, overexpressed human HO-1 mRNA in all organs. Transgene-derived HO-1 was detectable at the protein level only in the thymus. Thymocytes from transgenic rats displayed cell subsets and allogeneic proliferative responses in vitro comparable to those of nontransgenic animals. HO-1 transgenic rats used as recipients rejected cardiac allografts with the same kinetics as control rats. After immunization, transgenic rats showed normal antibody and proliferative responses against cognate antigens.

Materials and Methods

DNA Construct.
The backbone of the construct (pCombi) used to generate transgenic rats with tet controllable expression was kindly provided by Dr. U. Certa (Hoffmann-La Roche, Basel, Switzerland) and has been used previously to generate transgenic mice (14). In the construct used to generate HO-1 transgenic rats (pCombiHO-1; Fig. 1Go), the mouse ubiquitous H-2Kb promoter linked to an enhancer from the murine T cell receptor {alpha} chain gene (13) and a hybrid intron were inserted 5' of the coding sequence for the tTA and SV40 polyadenylation (polyA) sequences of pCombi. In the absence of tet, the tTA binds to the tet operator (tetOP) and, in conjunction with a CMV minimal promoter (pCMVmin), activates transcription of an expression cassette containing (from the 5' end to the 3' end) a ß globin intron, the human HO-1 cDNA (kindly provided by S. Shibahara, Tohoku University School of Medicine, Sendai, Japan) in which a FLAG sequence was inserted in its 3' end, and SV40 polyA sequences.



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  		Figure 1. pCombiHO-1 DNA construct used for the generation of HO-1 transgenic rats. The ubiquitous H-2Kb promoter drives the expression of the tet transactivator (tTA). In the absence of tetracycline (tet), tTA binds to the tet operator (tetOP) and with a CMV minimal promoter (pCMVmin), activates transcription of an expression cassette containing a ß globin intron, the human HO-1 cDNA with a FLAG sequence in its 3' end and SV40 polyA sequences.

 
Generation of HO-1 Transgenic Rats.
Transgenic rats were generated by pronuclear microinjection of Sprague–Dawley eggs (IFFA-CREDO, L’Arbresle, France) with the SalI-BamHI fragment of pCombiHO-1 (9 kb) using procedures previously described (15, 16). To inhibit transgene expression, tet (1 mg/ml) and sucrose (36 mg/ml) were included in the drinking water (protected from light) of surrogate mothers and litters derived from microinjection. Analysis of transgene expression was performed at least 10 days after tet withdrawal. Transgenic rats were first identified by analysis of tail DNA by polymerase chain reaction (PCR) using human HO-1 primers: sense; 5'-GTC TTC GCC CCT GTC TAC TT-3' and antisense; 5'-CTC TTC TAT CAC CCT CTG CCT-3'. PCR was also performed in parallel for the rat reference gene hypoxanthine phosphoribosyltransferase using the following primers: sense; 5'-GCG AAA GTG GAA AAG CCA AGT-3'; antisense; 5'-GCC ACA TCA ACA GGA CTC TTG TAG-3'. Both PCR reactions consisted of 5 min at 94°C, 5 min at 62°C and 30 cycles of 72°C for 30 sec, 60°C for 30 sec and 94°C for 30 sec. Positive animals were then confirmed by Southern blot analysis of DNA digested with EcoRV using a 32P-labeled dCTP fragment of pCombiHO-1. Determination of zygosity in HO-1 transgenic animals was performed using a new real-time PCR-based method (17) using an ABI Prism 7700 Sequence Detector System (PE Applied Biosystems, Foster City, CA).

RNA Extraction and Real-Time Quantitative PCR.
Total RNA was isolated using Trizol (Life Technologies, Paris, France), treated with DNAse (Roche, Indianapolis, IN) and reversed transcribed (Life Technologies, Paris, France) into cDNA. Real-time quantitative PCR was performed using primers for human HO-1: forward; CTC AAC ATC CAG CTC TTT GAG GAG TTG CAG G-3' and reverse; 5'-TGG GAG CGG GTG TTG AGT-3' and a labeled TaqMan® probe 5'FAM-CTC AAC ATC CAG CTC TTT GAG GAG TTG CAG G-TAMRA3'. Rat hypoxanthine phosphoribosyltransferase was amplified using the same primers described above and a labeled TaqMan® probe 5'VIC-CAA AGC CTA AAA GAC AGC GGC AAG TTG AAT-TAMRA3'. Transcript levels were calculated according to the 2-{Delta}{Delta}Ct method (18).

Analysis of HO-1 Expression.
Immunohistological analysis was performed on cryostat sections using a rabbit anti-HO-1 antibody (reacting with both human and rat HO-1) (Stressgen, Victoria, British Columbia, Canada) using techniques previously described (7). HO-1 enzymatic activity was analyzed in the microsomal fraction of the thymus as previously described (19).

Keyhole Limpet Hemocyanin (KLH) Immunization.
KLH (Sigma, St Louis, MO) was injected in the footpad as previously described (20). Anti-KLH antibodies were detected in sera using an enzyme-linked immunosorbent assay and proliferative responses against KLH were analyzed using popliteal lymph node cells 10 days after immunization as previously described (20).

Mixed Leukocyte Reaction (MLR).
Splenic or thymic cells from non-transgenic or HO-1 transgenic rats (MHC haplotype RT1u) were cultured with {gamma}-irradiated allogeneic antigen presenting cells (APCs) from LEW.1A rats (MHC haplotype RT1a) with or without recombinant IL-2 (100 U/ml) as previously described (20).

Cytofluorimetry.
Cells were incubated with mAbs for 20 min at 4°C, washed twice, and analyzed using a FACScalibur (Becton Dickinson, Mountain View, CA). The following mouse anti-rat mAbs obtained from the European Collection of Cell Culture (Salisbury, UK) were used after coupling to FITC, biotin, or PE (Bioatlantic, Nantes, France): OX6 (MHC class II, B cells, macrophages and activated T cells), W3/25 (CD4; mainly CD4+ T cells), OX7 (Thy-1.1; CD90, thymocytes), OX33 (CD45RB; B cells), OX42 (CD11b and CD11c; macrophages and dendritic cells), OX8 (CD8; CD8{alpha} + T cells), OX39 (CD25; activated T cells), OX22 (CD45RC), and OX85 (CD62L; naive T cells). The JJ319 MAb (CD28; T cells) was kindly provided by Dr. T. Hüning (University of Würzburg, Germany). The FITC-conjugated anti-CD3 (clone G4.18; T cells), anti-B7-2 (CD86; macrophages and dendritic cells), and PE-conjugated anti-B7.1 (CD80; macrophages and dendritic cells) mAbs were purchased from PharMingen (San Diego, CA). The FITC anti-CD161a (NKR-P1A, NK cells) was purchased from Serotec (Oxford, UK).

Heart Transplantation.
Cardiac allografts from LEW.1A donors were placed into the abdomen of HO-1-transgenic rats and graft survival was monitored daily by palpation through the abdominal wall. Rejection was defined as cessation of cardiac beating.

Histopathological Analyses.
Hematoxylin and eosin-stained tissue sections of paraffin-embedded samples of the indicated organs were analyzed by an experienced pathologist (C.T.).

Results

Generation of HO-1 Transgenic Rats with the Tet-Off System.
After transient transfection of COS cells with pCombiHO-1, expression of HO-1 was undetectable in the presence of tet (2 µg/ml) and was induced in its absence, as analyzed by Western blot and enzymatic assays in cellular lysates (data not shown).

Immediately after microinjection of the 9-kb fragment of pCombiHO-1, 702 one-cell embryos were transferred into foster mothers and 111 pups were obtained. Eight founders (7.2% of newborns) were identified by PCR analysis and confirmed by Southern blot analysis as carriers of the entire HO-1 transgene integrated in a single-site (data not shown). The frequency of transgenic rats obtained with pCombiHO-1 was comparable to that obtained previously for other transgenes (16, 21, 22). Four founders transmitted the transgene to their descendants and transgenic lines were derived from each of them.

Analysis of Transgene-Derived HO-1 mRNA.
Expression of transgene-derived HO-1 mRNA was analyzed using real-time reverse transcription PCR in organs from rats continuously kept with tet or withdrawn from tet for 10 days. Two lines among 4, 12.4 (Fig. 2AGo) and 12.6 (Fig. 2BGo), expressed transgene-derived HO-1 mRNA, mainly in lymphoid organs and the highest levels being observed in the thymus, as previously described in transgenic mice generated with the H-2Kb promoter (13). HO-1 mRNA increased in both lines in the absence of tet. Line 12.4 expressed higher levels in the absence and lower levels in the presence of tet than line 12.6. Low levels of HO-1 mRNA were observed in the heart and kidney in line 12.4 but not in line 12.6. The liver did not show HO-1 mRNA accumulation in neither line.



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  		Figure 2. Quantification of hHO-1 mRNA. Real-time quantitative RT-PCR was used to analyze hHO-1 in the indicated organs from (A) line 12.4 and (B) line 12.6 kept with or without tetracycline (tet) for 10 days. Results from one animal in each condition are representative of two to three animals analyzed in each line.

 
Analysis of Transgene-Derived HO-1 Protein Expression.
Homozygous HO-1 transgenic rats from lines 12.4 and 12.6 were continuously kept with tet or withdrawn from tet for 10 days and their organs were analyzed for HO-1 protein expression. As observed by immunohistological analysis (Fig. 3Go), low levels of endogenous HO-1 were detected in the thymus of control rats and lines 12.4 and 12.6 expressed higher levels. Cells expressing HO-1 in transgenic rats were scattered in the cortex and medulla. Higher expression of HO-1 was confirmed by Western blot analysis in the thymuses of HO-1 transgenic rats versus controls (data not shown).



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  		Figure 3. Thymic HO-1 expression. Homozygous HO-1 transgenic rats were maintained without tet for 10 days and HO-1 expression was analyzed on thymus cryostat section by immunohistology using an anti-HO-1 antibody (objective x20). Results from one animal for each condition are representative of two to three animals analyzed in each line.

 
Analysis of HO-1 enzymatic activity showed increased levels in the thymus in lines 12.6 and 12.4 versus controls (2 and 1.49, respectively vs 0.3 nmol/mg/h). Transgene-derived HO-1 was undetectable in the lymph nodes, spleens, livers, kidneys, and hearts of these animals, as analyzed by immunohistology and Western blot (data not shown).

Thymic Cell Subsets and Immune Responses Were Unmodified in HO-1-Transgenic Rats.
Histopathological analysis of the thymus, lymph nodes, spleen, liver, kidney, intestine, lung, and heart in lines 12.4 and 12.6 after tet withdrawal revealed no anomalies (data not shown). Analysis by cytofluorimetry of the major leukocyte subsets (CD4+, CD8+, B cells, macrophages, CD90+ thymocytes, and NK cells) of lines 12.6 (Fig. 4AGo) and 12.4 (data not shown) revealed no differences compared with controls of the same age. Other markers, such as CD25, CD45RC, CD62L, CD86, and CD80, did not reveal any differences in the thymic cells of transgenic versus nontransgenic rats. The same analysis performed on spleen cells from transgenic lines 12.4 and 12.6 did not show any phenotypic differences compared with controls (data not shown).



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  		Figure 4. Thymic subsets and immune responses. Nontransgenic litter mates or homozygous HO-1 transgenic rats from line 12.6 maintained with or without tet for 10 days were analyzed for phenotype and function of their immune cells. (A) Thymic cells analyzed by cytofluorimetry. Numbers within each window represent the percentage of positive cells. (B) Proliferation of thymic cells cultured with medium alone (stippled histogram) or with allogeneic APCs (LEW.1A) in the absence (white histogram) or presence (black histogram) of IL-2 (100 U/ml). [3H] thymidine incorporation (mean ± SD of triplicate cultures) was evaluated after 5 days of culture. Nontransgenic (Non Tg) or transgenic (Tg) rats of line 12.6 were kept with or without tet (+ Tet or – Tet, respectively) for 10 days, immunized or not with KLH in the footpad and analyzed 10 days later. (C) Draining lymph node cells were removed and proliferation against different concentrations of KLH was tested in vitro. [3H] thymidine incorporation (mean ± SD of triplicate cultures) was evaluated after 3 days of culture. Each curve represents the proliferation of cells from a single animal. (D) Anti-KLH antibodies were detected in sera of rats of line 12.6 using an ELISA. The results for the KLH experiments were an average of two animals from each condition. Results are expressed as optical density (OD).

 
Analysis of allogeneic proliferation of thymic cells from transgenic rats (haplotype RT1u) against APCs from LEW.1A (haplotype RT1a) rats showed strong proliferation for lines 12.6 (Fig. 4BGo) and 12.4 (data not shown), which were comparable to those of control nontransgenic rats. The same MLRs performed with spleen cells from transgenic lines 12.4 and 12.6 did not show any differences compared with controls (data not shown).

To evaluate the immune responses of transgenic rats against cognate antigens, the animals were continuously kept with tet or withdrawn from tet for 10 days. KLH was subsequently injected into the footpad and 10 days later the analyses of proliferative responses of draining lymph nodes cells (Fig. 4CGo) and sera anti-KLH antibody levels (Fig. 4DGo) in HO-1-transgenic rats showed responses similar to those of controls.

Allograft Survival Was Not Prolonged in HO-1-Transgenic Rats.
We used transgenic rats of both lines as recipients of cardiac allografts from LEW.1A donors. Rejection of LEW.1A heart by non-transgenic controls (6.2 ± 0.8, n = 3) was indistinguishable from those of transgenic recipients (line 12.6, 6 days, n = 3; line 12.4, 7 days, n = 3).

Discussion

We generated rats transgenic for HO-1, which overexpressed transgene-derived HO-1 protein in the thymus. The H-2Kb promoter was used because of the fact that in transgenic mice, it has been shown to drive expression of transgenes primarily in leukocytes and endothelial cells (13). Overall, the expression levels of HO-1 obtained in the transgenic rats was low because its expression was only detectable in two of four lines and transgene-derived HO-1 protein was only detectable in the thymus. Although transgenic pigs have also been generated using the same promoter (23), we cannot formally exclude the possibility that species restrictions may exist in the rat for high expression of transgenes placed under its transcriptional control. Alternatively, as is the case for every promoter lacking insulating sequences, its activity may be largely dependent on the chromatin configuration at the point of insertion of the transgene into the genome.

The tet-off system integrated into a single DNA construct used in this study has been previously applied to generate lines of transgenic mice, from which a proportion showed tet-controlled expression of the transgene (14). Both rat transgenic lines showed higher HO-1 expression in the thymus in the absence than in the presence of tet at the mRNA level suggesting regulation of HO-1 expression.

Among other observable protective effects, mice transgenic for HO-1 have shown decreased inflammation after oxidative injury (8, 10–12), but the effect of HO-1 in other inflammatory models has not been analyzed in transgenic animals. In our study, rats transgenic for HO-1 displayed thymic and splenic cell subsets as well as cellular and humoral anti-KLH responses comparable with those of nontransgenic rats. In MLRs, thymocytes and splenocytes from HO-1 transgenic rats showed proliferative responses of the same magnitude as control rats. Moreover, in a cardiac allograft model, HO-1 transgenic animals rejected allogeneic hearts with the same kinetics as controls. These results suggest that rats transgenic for HO-1 show normal differentiation of immune cells and immune responses after a relative short-term (10 d) induction of HO-1 expression. Nevertheless, the low HO-1 expression observed in secondary lymphoid organs do not allow us to conclude that HO-1 overexpression does not affect immune responses. New transgenic animals with high HO-1 expression in peripheral lymphoid organs are currently being generated. Thymic cells displayed clear HO-1 protein overexpression, and some important cell processes (apoptosis, cytokine production) have not yet been analyzed. Furthermore, overexpression during the whole life of these transgenic rats may modify thymocyte differentiation. The generation of transgenic rodents with expression of HO-1 by cells of the immune system will represent an important tool for the analysis of HO-1 function.

Acknowledgments

We are grateful to Helga Smit, Claire Usal, Emmanuel Merieau, and Bernard Martinet for heart transplantations and animal care as well as researchers who contributed reagents.

Footnotes

This work was financed in part by the Fondation Transvie and the European Union Grant QLK3-CT2001-00422.

1 Both authors share senior authorship. Back

2 To whom requests for reprints should be addressed at INSERM U437, 30 Bd Jean Monnet, 44093 Nantes, France. E-mail: ianegon{at}nantes.inserm.fr Back

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