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Cardiovascular Research 1999 42(3):670-679; doi:10.1016/S0008-6363(99)00007-3
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Copyright © 1999, European Society of Cardiology

The role of Bcl6 in mature cardiac myocytes

Takehiko Yoshidaa,e, Tetsuya Fukudaa,g, Masahiko Hatanoa, Haruhiko Kosekib, Shin-ichiro Okabea, Kazuki Ishibashia, Satoko Kojimaa, Masafumi Arimaa, Issei Komurof, Genichiro Ishiid, Tohru Mikig, Shinsaku Hirosawag, Nobuyuki Miyasakag, Masaru Taniguchic, Takenori Ochiaie, Kaichi Isonoe and Takeshi Tokuhisaa,*

aDepartment of Developmental Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan
bDepartment of Molecular Embryology, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan
cDepartment of Molecular Immunology, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan
dDepartment of Pathology,Chiba University Graduate School of Medicine, Chiba 260-8670, Japan
eSecond Department of Surgery, Chiba University School of Medicine, Chiba 260-8670, Japan
fDepartment of Medicine III, The University of Tokyo School of Medicine, Tokyo 113, Japan
gFirst Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo 113, Japan

tokuhisa{at}med.m.chiba-u.ac.jp

* Corresponding author. Tel.: +81-43-226-2181; fax: +81-43-226-2183

Received 26 June 1998; accepted 25 November 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objective: The Bcl6 gene encodes a sequence-specific transcriptional repressor and is ubiquitously expressed in adult murine tissues including heart muscle. The objective of this study was to examine the role of Bcl6 in cardiac myocytes. Method: We developed Bcl6-deficient (Bcl6–/–) mice and histologically examined hearts from these mice. Results: Massive myocarditis with eosinophilic infiltration occurred in Bcl6–/– mice after 4–6 weeks of age. Since expression of the Bcl6 gene was induced in normal cardiac myocytes after 2 weeks of age and thereafter detected through adulthood, loss of Bcl6 in mature cardiac myocytes may be related to the induction of eosinophilic myocarditis. To examine the effects of eosinophils from Bcl6–/– mice on normal hearts, bone marrow cells from Bcl6–/– mice were adoptively transferred into sublethally irradiated RAG1-deficient mice. Although massive eosinophilic infiltration was detected in conjunctivas and spleens from the chimeric mice, myocarditis was never observed. Electron microscopic analysis of cardiac myocytes from Bcl6–/– mice revealed a spectrum of degenerative changes prior to eosinophilic infiltration. Conclusion: Bcl6 may not be essential for the maturation of cardiac myocytes but may play a role in protecting mature cardiac myocytes from eosinophilic inflammation.

KEYWORDS Bcl6; Knockout mice; Myocaditis; Terminal differentiation; Tissue eosinophilia


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
The Bcl6 gene was identified from the translocation breakpoint (3q27) in lymphomas [1–4]. The Bcl6 gene is ubiquitously expressed in adult human tissues, especially in skeletal muscle [4] and germinal center B cells [5,6]. Since the Bcl6 gene encodes a protein containing Krüppel-type zinc finger motifs, it may function as a sequence-specific transcriptional factor. Recent in vitro studies have suggested that this gene acts as a transcriptional repressor [7–9]. However, physiological functions of this protein have remained unknown. We recently cloned the murine homologue gene, the amino acid sequence of which is 95% identical to that of human Bcl6 [10]. The expression was also ubiquitously detected in adult murine tissues including heart muscle and was transiently upregulated in activated lymphocytes as an immediate early gene. It was also progressively upregulated in differentiating keratinocytes with cell cycle arrest [11]. Therefore, Bcl6 plays a role not only in activated lymphocytes but also in mature tissues at terminal differentiation stages.

To examine the physiological functions of Bcl6, the gene was disrupted in the mouse germ line [12–14]. In homozygous mutant (Bcl6–/–) mice there was an inflammatory response in multiple organs that was characterized by infiltration of eosinophils. Many factors are involved in the generation of tissue eosinophilia. Interleukin-5 (IL-5) produced by T helper cell type 2 (Th2) is an eosinophil growth factor and is also important for eosinophil hematopoiesis and for survival [15,16]. Since the increased expression of Th2 type cytokines including IL-5 is induced in T cells from Bcl6–/– mice by anti-CD3 activation, mechanisms of this eosinophilic inflammation could be partly explained by functional dominance of Th2 in Bcl6–/– mice [12,13].

The Bcl6–/– mice we developed also had a massive myocarditis with eosinophilic infiltration. Eosinophils are potent pro-inflammatory cells involved in the pathogenesis of several human disorders such as asthma and chronic parasitic infection [17], and eosinophil granule proteins are toxic for cardiac myocytes [18]. Thus, eosinophils might initially infiltrate to injure heart muscle in these Bcl6–/– mice. Since expression of the Bcl6 gene was detected in normal hearts from adult mice [10], loss of Bcl6 expression in cardiac myocytes may be related to the induction of eosinophilic myocarditis. However, a search of the literature revealed no detailed analysis of the relation between loss of the expression in cardiac myocytes and induction of the eosinophilic myocarditis in Bcl6–/– mice. We show here that eosinophils from Bcl6–/– mice did not induce eosinophilic myocarditis in sublethally irradiated RAG1-deficient mice with normal hearts. The role of Bcl6 in protecting mature cardiac myocytes from eosinophilic infiltration is discussed.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Gene targeting in embryonic stem cells
A targeting vector was generated from a 16-kb genomic clone containing the Bcl6 gene isolated from a genomic DNA library of mouse strain 129/SV. A 4.0-kb fragment from the XhoI site in exon 3 to the EcoRI site in exon 6 was replaced by a neomycin resistant gene cassette (pMC1-neo) in reverse orientation relative to Bcl6 transcription (Fig. 1A). For negative selection, a herpes simplex thymidine kinase gene cassette was fused at the 5' end of the genomic clone in the same orientation relative to Bcl6 transcription.


Figure 1
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  		Fig. 1 Targeted disruption of the Bcl6 gene. (A) The Bcl6 locus containing eight exons (closed boxes) and a portion of 5' and 3' flanking regions is shown (top). The targeting vector (middle) was designed to replace a DNA segment through exon 3 to exon 6 by a neomycin resistant gene cassette (NEO). A herpes simplex thymidine kinase gene cassette is indicated as TK. The resulting targeted allele is depicted in the bottom panel. Restriction enzyme abbreviations; A, ApaI; E, EcoRI; X, XhoI. (B) Southern blot analysis of genomic DNA digested with ApaI. A probe used for Southern screening is indicated as Probe in (A). The position of the hybridizing fragments for the wild type (6.7 kb) and the targeted allele (5 kb) is shown. (C) Northern blot analysis of Bcl6 mRNA in kidneys from four-week-old mice. The probe revealed the authentic mRNA (3.8 kb) prominent in Bcl6+/+, diminished in Bcl6+/–, and undetectable in Bcl6–/– mice. The ethidium bromide-stained gel, prior to transfer, is shown below for normalization of RNA loading.

 
R1 embryonic stem cells were transfected with the linearized targeting vector by electroporation and subjected to positive and negative selection using G418 and gancyclovir for 14 days. Double resistant colonies were picked up, replated individually and subjected to genotype analysis by Southern blots. Two independent targeted clones were used to generate chimeric mice using the aggregation method [19] with slight modification. Tail DNAs from agouti pups obtained from mating with C57BL/6 mice were analyzed by Southern blots. Homozygous mutant pups were generated by intercrossing heterozygous mutant mice. Specific pathogen-free C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). All procedures conformed to Chiba University Resolution on Use of Animals in Research and were approved by the Institutional Animal Care and Use Committee at Chiba University School of Medicine.

2.2 Southern blot analysis
Southern blot analysis was done as described [20]. Briefly, genomic DNA isolated from the mutant offspring was digested with ApaI, separated on a 1% agarose gel, and transferred to a nylon membrane (Amersham International) and fixed by cross-linking with UV irradiation and by baking at 120°C for 1 h. The filter was pre-hybridized for 3 h and hybridized overnight at 42°C in 50% formamide hybridization buffer with 0.5% SDS, 1% blocking reagent and 15 ng/ml of probe. Following hybridization, the filter was washed twice for 5 min with 2X SSC and 0.1% SDS at room temperature and twice for 15 min with 0.1X SSC and 0.1% SDS at 55°C. The probe was detected with sheep anti-digoxigenin (DIG) antibody conjugated with alkaline phosphatase (Boehringer Mannheim, Mannheim, Germany). The antibody detection reaction was performed using an enhanced chemiluminescent detection system (Boehringer Mannheim). As a probe, a 0.75-kb XbaI–PstI fragment, which is external to the targeting vector, was subcloned into a pGEM vector (Promega, Madison, WI) and labeled with DIG (Boehringer Mannheim) by polymerase chain reaction, using SP6 and T7 primers. The probe detected the wild type allele as a 6.7-kb fragment and the mutant allele as a 5.0-kb fragment (Fig. 1B).

2.3 Northern blot analysis
Total RNAs were extracted from adult female mouse tissues with the Trizol reagent (Life Technologies, Gaithersburg, MD). Northern blot analysis was done as described [10]. Briefly, total RNAs (20 µg) were electrophoresed through a 1.0% agarose gel containing formaldehyde, transferred to a nylon membrane. The filter was hybridized with a DIG-labeled probe and followed by the method described for Southern blot analysis. A 643 bp ApaI fragment (844–1487 bp) of the Bcl6 cDNA was used as a probe. This ApaI fragment was subcloned into a pGEM vector and labeled by DIG using PCR with T7 and SP6 primers. A probe for {alpha}-cardiac myosin heavy chain ({alpha}MHC) mRNA was prepared by labeling a synthetic oligomer (5'-CCTCTCCAGCAGACCCTCGCTGTGGCCAATCCACAAT-AAACATAAACGTTCG-3') with DIG at the 3' end by a 3' end-labeling kit (Boehringer Mannheim).

2.4 Histopathological preparations
Organs were dissected from mice (4–12 weeks-old) and fixed in 10% buffered formalin. The tissues were processed through paraffin embedding using standard procedures. A transverse section of heart was made at about 1/3 of total length from the apex. Sections (4 µm) were stained with hematoxylin and eosin. Eosinophils in tissues were identified by Luna staining [21]. For immunohistochemistry, animals (4 week-old) were perfused with a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Organs were dissected and postfixed with 4% paraformaldehyde for 12 h. The tissues were equilibrated with 20% sucrose, sectioned at 10 µm on a cryostat. Sections were incubated overnight with anti-vinculin antibody (Sigma Chemical, St. Louis, MO) at 4°C, followed by biotin-conjugated antibody to mouse immunoglobulin (Ig; Nichirei, Tokyo, Japan) for 2 h at room temperature. StreptABComplex/HRP (Dako, Carpinteria, CA) was used as the third-phase reagent. Bound horseradish peroxidase (HRP) was visualized using a DAB kit (Nichirei).

2.5 Electron-microscopic analysis
Hearts from mice under deep ether anesthesia were perfused with a mixture of 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The hearts were excised and promptly fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3). The ventricular portion of the heart was post-fixed in 1% OsO4 for 2 h, dehydrated in an ascending series of ethanol solutions, passed through propylene oxide, and embedded in epoxy resin. Finally, ultrathin sections were prepared using a Porter-Blum ultramicrotome, and doubly stained with uranyl acetate and lead citrate. The sections were viewed under a JEOL 1200EX electron microscope.

2.6 Generation of bone marrow (BM) chimeras
Generation of BM chimeras was done as described [14]. Briefly, RAG1-deficient mice (8–16 weeks-old) were sublethally irradiated (3.5 Gy) and injected with 2–5x106 BM cells from Bcl6–/– (Bcl6–/–RM) or Bcl6+/+ (Bcl6+/+RM) littermates (3–5 weeks-old). Since mature lymphocytes did not develop in RAG1-deficient mice, the reconstitution was confirmed 8–12 weeks after transplantation by identifying CD3+ and B220+ mature lymphocytes in peripheral blood from Bcl6–/–RM and Bcl6+/+RM using a flow cytometry.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 Generation of Bcl6 knockout mice
The murine Bcl6 gene was mutated in embryonic stem cells by homologous recombination (Fig. 1A). A genomic DNA segment between exon 3 through exon 6 which encodes the zinc finger motifs of Bcl6 was replaced by a neomycin resistant gene cassette. This procedure led to production of a fusion mRNA (expected size; 4.3 kb) whose size is larger than that of authentic Bcl6 mRNA (3.8 kb). Heterozygotes (Bcl6+/–) were interbred, and their progeny were genotyped by Southern blot analysis (Fig. 1B). The number of each genotype from embryos (day 14.5) and newborn mice exhibited a Mendelian distribution. However, the number of homozygous mutant (Bcl6–/–) mice that lived longer than 3 weeks was smaller than that expected; 20 (Bcl6–/–), 123 (Bcl6+/–), and 62 (Bcl6+/+), indicating some postnatal loss of Bcl6–/– mice. Neither the authentic Bcl6 mRNA nor the fusion mRNA (4.3 Kb) was detected in total RNA from any tissues of Bcl6–/– mice examined by Northern blot analysis (Fig. 1C).

Viable Bcl6–/– mice were indistinguishable from Bcl6+/– and Bcl6+/+ littermates at birth. At approximately 3 weeks of age, Bcl6–/– mice could be clearly identified by their small size. These features became more prominent as the mice aged. The mean body weight of Bcl6–/– mice was significantly less than that of Bcl6+/– and Bcl6+/+ littermates after 3 weeks of age (Fig. 2).


Figure 2
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  		Fig. 2 Growth retardation of Bcl6–/– mice. The mean body weight±SD of Bcl6–/– (open circle), Bcl6+/– (closed circle) and Bcl6+/+ (closed triangle) mice were calculated from 10–20 littermates.

 
3.2 Massive myocarditis with eosinophilic infiltration in Bcl6–/– mice
Bcl6–/– mice appeared to be sick from 4–6 weeks after birth and most (18/20) died by 9 weeks after birth. At necropsy, their hearts were markedly enlarged with ventricular dilatation and ventricular wall thinning (Fig. 3A). Light-microscopic examinations showed severe degeneration of myofibers, an increase in interstitial fibrosis and massive infiltration of granulocytes (Fig. 3B). More than 50% of those granulocytes were eosinophils determined by Luna staining (Fig. 4). Inflammation with eosinophilic infiltration was also observed in lungs and spleens from Bcl6–/– mice (Fig. 4). When Bcl6–/– mice (n=5) between 6 and 12 weeks of age were histopathologically examined, inflammation with eosinophilic infiltration was occasionally observed in heart (4/5), spleen (4/5), lung (2/5), and liver (1/5), but not in kidney (0/5). However, none of Bcl6–/– mice examined showed eosinophilia in peripheral blood. Numbers of eosinophils in peripheral blood from Bcl6–/–, Bcl6+/–, and Bcl6+/+ mice (n=3) were 53±50/µl, 110±98/µl, and 190±99/µl, respectively.


Figure 3
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  		Fig. 3 Massive myocarditis with eosinophilic infiltration in Bcl6–/– mice. (A) Cross section of hearts from 6 week-old Bcl6–/– (KO) and Bcl6+/+ (WT) littermates showing left (LV) and right (RV) ventricles. The cavity of the left ventricle of Bcl6–/– mice is enlarged and the wall is thinner than that of Bcl6+/+ mice. (B) Hematoxylin and eosin staining of the left ventricle of Bcl6–/– (KO) and Bcl6+/+ (WT) littermates. The ventricle of KO mouse shows severe degeneration of myocytes and massive granulocyte infiltration (x100).

 

Figure 4
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  		Fig. 4 Inflammation with eosinophilic infiltration in various organs from Bcl6–/– mice. Hematoxylin and eosin staining of heart (A, B), lung (D, E), and spleen (G, H) from Bcl6–/– mice at 6 weeks of age demonstrated inflammation with granulocyte infiltration. B, E, and H (x100) were the extended picture of the marked areas in A, D, and G (x20), respectively. Luna staining of the heart (C), the lung (F), and the spleen (I) (x100).

 
3.3 Condition of cardiac myocytes from Bcl6–/– mice before infiltration of eosinophils
Since expression of Bcl6 mRNA was detected in normal hearts from adult mice [10], Bcl6 may play a role in mature cardiac myocytes. Thus, we examined the condition of hearts from Bcl6–/– mice prior to the infiltration of eosinophils. Whenever we histologically examined hearts from Bcl6–/– mice younger than 4 weeks of age, cardiac myocytes from Bcl6–/– mice appeared normal. Thus, we analyzed the maturity of cardiac myocytes from Bcl6–/– mice at 4 weeks of age. Maturation of cardiac myocytes is synchronous with alteration of two isoforms of MHC, {alpha}MHC and βMHC, in ventricular myocytes. {alpha}MHC mRNA increases in ventricular myocytes of embryos from day 17 and finally replaces βMHC mRNA to develop into a mature stage in the first postnatal week [22]. Northern blots were prepared to determine expression of Bcl6 and {alpha}MHC mRNA in ventricular myocytes of normal mice during development and of Bcl6–/– mice at 4 week-old before infiltration of eosinophils (Fig. 5). Bcl6 mRNA was not detected in normal ventricular myocytes until 1 week after birth but was induced at 2 weeks of age and thereafter detected through adulthood. The plateau level of {alpha}MHC mRNA was evident in normal ventricular myocytes after birth. Although Bcl6 mRNA was not detected in ventricular myocytes of Bcl6–/– mice, the level of {alpha}MHC mRNA in the myocytes was similar to that in the normal myocytes. Thus, maturation of cardiac myocytes from Bcl6–/– mice seemed to be normal.


Figure 5
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  		Fig. 5 Expression of Bcl6 mRNA in ventricular myocytes. Total RNAs were isolated from ventricular muscle without cell infiltration of Bcl6+/+ and Bcl6–/– mice. Expression of Bcl6 and {alpha}MHC mRNA was analyzed by Northern blot. The ethidium bromide-stained gel, prior to transfer, is shown below for normalization of RNA loading.

 
3.4 Relation between loss of Bcl6 expression in cardiac myocytes and induction of myocarditis with eosinophilic infiltration in Bcl6–/– mice
To examine the relation between loss of Bcl6 expression in mature cardiac myocytes and the induction of myocarditis, BM cells from Bcl6–/– mice were transferred into sublethally irradiated RAG1-deficient mice (Bcl6–/–RM) with normal hearts. Flow cytometric analysis of thymocytes and spleen cells from Bcl6–/–RM 3 months after transplantation revealed normal development of mature lymphocytes derived from BM cells of Bcl6–/– mice [14]. Although eosinophilic infiltration was observed in conjunctiva and spleen from approximately 50% of Bcl6–/–RM (7/15) (Fig. 6) but not in these tissues from Bcl6+/+RM (0/15), cell infiltration was not detected in any heart from Bcl6–/–RM examined (0/15). These findings in Bcl6–/–RM suggest a protective role for Bcl6 in induction of eosinophilic myocarditis.


Figure 6
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  		Fig. 6 Eosinophilic infiltration in conjunctiva from Bcl6–/–RM. Bcl6–/–RM displayed conjunctivitis 3 months after transplantation. Hematoxylin and eosin staining of conjunctiva (A) and spleen (C) from Bcl6–/–RM demonstrated inflammation with granulocyte infiltration. (x20) Luna staining of the conjunctiva (B) and the spleen (D) (x100).

 
We also examined the role for Bcl6 in mature cardiac myocytes from Bcl6–/– mice prior to the infiltration of eosinophils by histological examination of the myocytes. When we examined cardiac myocytes from Bcl6–/– mice at 4 weeks of age without eosinophilic infiltration by a microscope, the myocytes were histologically normal by hematoxylin–eosin staining (Fig. 7A, B) and by immunohistochemical analysis using anti-vinculins (Fig. 7C, D) or anti-dystrophin antibodies (data not shown). However, electron-microscopic analysis of those Bcl6–/– myocytes revealed a variety of degenerative changes such as an irregular size of condensed mitochondria within an electron-lucent cytoplasm and sparse organelle (Fig. 8). These results suggest that cardiac myocytes without Bcl6 are intrinsically damaged prior to eosinophilic infiltration.


Figure 7
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  		Fig. 7 Immunohistochemistry of Bcl6–/– heart prior to eosinophilic infiltration. The left ventricles from Bcl6–/– (KO) and Bcl6+/+ (WT) littermates at 4 weeks of age were stained with hematoxylin and eosin (A, B), or anti-vinculin antibody (C, D). Hematoxylin counterstain (C, D) (x200).

 

Figure 8
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  		Fig. 8 Transmission electron micrographs of ventricular myocytes of Bcl6–/– heart prior to eosinophilic infiltration. The left ventricles of Bcl6–/– (KO) and Bcl6+/+ (WT) littermates at 4 weeks of age were examined using an electron-microscope. Ventricular myocyte of Bcl6+/+ littermate (A) and Bcl6–/– mouse (B) at 6 weeks of age. (x5,000) (B) reveals a variety of degenerative changes such as condensed mitochondria within electron-lucent cytoplasm. Mitochondrial ultrastructure in the Bcl6+/+ myocyte (C) and the Bcl6–/– myocyte (D) (x15 000).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
Massive myocarditis with eosinophilic infiltration was observed in Bcl6–/– mice after 4 weeks of age (Fig 3). Since the major infiltrates were eosinophils and eosinophil granule proteins mediate endomyocardial damage [18], it was suggested that infiltration of eosinophils is the initial event of myocarditis in these mice. However, Bcl6–/–RM did not develop myocarditis even though eosinophilic infiltration was observed in conjunctiva and spleen from approximately 50% of Bcl6–/–RM (Fig 6). Thus, eosinophils from Bcl6–/– mice may not be sufficient to develop the myocarditis. This notion is supported by evidence that the IL-5 transgenic mice had hypereosinophilia in peripheral blood and infiltration of eosinophils in muscle, liver, lung, and gut, but no myocarditis [15,16]. In some cases of the idiopathic hypereosinophilic syndrome, pathological evidence showed that infiltration of eosinophils occurred as a secondary reaction to damaged tissues of unknown etiology [23,24]. Therefore, eosinophilic infiltration may not be the primary inflammatory reaction to cardiac myocytes from Bcl6–/– mice.

The Bcl6 gene was induced after 2 weeks of age in cardiac myocytes from normal mice but is probably not essential for the maturation of cardiac myocytes (Fig. 5). However, cardiac myocytes in Bcl6–/– mice after 4 weeks of age were degenerative with eosinophilic infiltration, thereby suggesting the relation between loss of Bcl6 expression and induction of the myocarditis. Since eosinophilic infiltration may not be the primary inflammatory reaction to cardiac myocytes from Bcl6–/– mice, Bcl6 in mature cardiac myocytes may play a role in protection from myocarditis. Indeed, electron-microscopic analysis of cardiac myocytes from Bcl6–/– mice without eosinophilic infiltration revealed a spectrum of degenerative changes including abnormal structure of mitochondria (Fig. 8). Those degenerative changes strongly suggested cell death of myocytes although the sarcolemma was well preserved. Therefore, degenerative changes of mature cardiac myocytes without eosinophilic infiltration may be the primary reaction leading to myocarditis in Bcl6–/– mice. These initial changes in hearts from Bcl6–/– mice are followed by eosinophilic infiltration. Since eosinophil granule proteins are toxic for cardiac myocytes [18], eosinophilic infiltration may accelerate heart injury in Bcl6–/– mice. However, myocardial ischemia may not be the major cause of myocardial injury in Bcl6–/– mice since degenerative changes were observed evenly in a large area of the heart but not so extensively in the endocardium where effects of ischemia are usually most prominent [25]. Further study is required to elucidate the precise cause of myocyte injury in Bcl6–/– mice.

Bcl6-deficient mice studied by other workers [12,13] also revealed an inflammatory response in multiple organs including heart, lung, liver, skin, conjunctiva, and gut characterized by infiltration of eosinophils. Inflammation in multiple organs including lung and heart with eosinophilic infiltration has been reported in the idiopathic hypereosinophilic syndrome [23,24,26], including the Loeffler’s syndrome [27]. Pathological findings of myocarditis in Bcl6–/– mice were similar to those in the human diseases such as the presence of mural thrombus [28,29], suggesting that Bcl6–/– mice can serve as an animal model for those human diseases. Mechanisms of tissue eosinophilia in those human diseases are unknown. Whereas eosinophil growth factors, such as IL–5, are involved in eosinophil hematopoiesis and survival, eosinophil adhesion and locomotion are predominantly controlled by chemoattractants. Eotaxin is the major eosinophil chemoattractant and induces a potent and rapid eosinophil-specific recruitment that is augmented by IL-5 [30,31]. Since the increased expression of Th2-type cytokines including IL–5 is induced in T cells from Bcl6–/– mice [12], overproduction of Th2 type cytokines may be a part of the etiology of tissue eosinophilia in Bcl6–/– mice. These studies may lead to elucidate mechanisms of tissue eosinophilia in the hypereosinophilic syndrome.

Although the Bcl6 gene is highly expressed in skeletal muscle at their terminal differentiation stage [10], we found no severe injury in skeletal muscle from Bcl6–/– mice. Mice lacking manganese superoxide dismutase [32] or transforming growth factor-β1 [33,34] demonstrated that the heart muscle is much more sensitive to stress than is the skeletal muscle in vivo. A recent in vitro study using a skeletal muscle cell line suggested a protective function for Bcl6 against myocyte cell death [35]. Thus, Bcl6 may be an essential molecule in mature myocytes to control protective mechanisms against specific stress, and this protective function may explain leukemogenesis of B cells with chromosomal translocation involving 3q27. In that case, Bcl6–/– mice may be useful for studying transcriptional regulatory mechanisms that allow for a flexible, adaptive response to various stresses.

Time for primary review 35 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
We thank Drs. I. Iwamoto, A. Mikata, M. Dezawa, S. Okada and K. Takahashi for discussion, T. Umemiya and Y. Iwata for skillful technical assistance, and E. Furusawa for secretarial assistance. This work was supported in part by the Grants-in-Aid for Cancer Research from the Ministry of Education, Science, Sports and Culture of Japan, the Grants from the Ministry of Health and Welfare of Japan, the Research Grant of the Princess Takamatsu Cancer Research Fund (#96-22807), and the Grant from Japan Cardiovascular Research Foundation.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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