Contribution of double-negative thymic precursors to CD8alpha alpha (+) intraepithelial lymphocytes of the gut in mice bearing TCR transgenes.

Using male and female RAG(-/-) mutant mice expressing TCR transgenes specific for MHC class I- or II-presented HY peptides, we performed quantitative and phenotypic comparisons between the TCR(+) lymphocytes present in the lymphoid organs and the gut mucosa in euthymic versus athymic (nude) animals. These comparisons suggest that only a minority of the TCR(+) CD8alpha alpha (+) intraepithelial lymphocytes (IEL) of the transgenic euthymic mice originate from hematopoietic precursors acquiring a TCR in the gut wall, while a majority of these CD8alpha alpha(+) IEL appear to be of thymic origin (as were all TCR(+) CD8alpha beta (+) or CD4(+) in any location); these last cells are released from the thymus as double-negative thymocytes, which are at a more immature stage (CD44(+)CD25(+)) in female mice than in males (CD44(-)). In view of previous observations that in non-transgenic athymic mice the CD8alpha alpha (+) TCR(+) IEL populations are also markedly reduced quantitatively, the possibility of a thymic contribution to these ontogenically peculiar populations may also exist in normal mice. At which stage of differentiation such precursors might leave the thymus of normal adult mice remains to be explored.

Lethal Mycobacterium bovis Bacillus Calmette Guérin infection in nitric oxide synthase 2-deficient mice: cell-mediated immunity requires nitric oxide synthase 2.

The role of nitric oxide (NO) in Mycobacterium bovis Bacillus Calmette Guerin (BCG) infection was investigated using nitric oxide synthase 2 (nos2)-deficient mice, because NO plays a pivotal protective role in M. tuberculosis infection. We demonstrate that nos2-deficient mice were unable to eliminate BCG and succumbed within 8 to 12 weeks to BCG infection (10(6) CFU) with cachexia and pneumonia, whereas all infected wild-type mice survived. The greatest mycobacterial loads were observed in lung and spleen. Nos2-deficient mice developed large granulomas consisting of macrophages and activated T cells and caseous necrotic lesions in spleen. The macrophages in granulomas from nos2-deficient mice had reduced acid phosphatase activities, suggesting that NO is required for macrophage activation. The absence of NOS2 affected the cytokine production of the Th1 type of immune response, except IL-18. Serum amounts of IL-12p40 were increased and IFN-gamma was decreased compared with wild-type mice. The lack of NOS2 resulted in an overproduction of TNF, observed throughout the infection period. Additionally, TNFR1 and TNFR2 shedding was altered compared with wild-type mice. Up-regulation of TNF may be compensatory for the lack of NOS2. The late neutralization of TNF by soluble TNF receptors resulted in heightened disease severity and accelerated death in nos2-deficient mice but had no effect in wild-type mice. In conclusion, the inability of nos2-deficient mice to kill M. bovis BCG resulted in an accumulation of mycobacteria with a dramatic activation of the immune system and overproduction of pro-inflammatory cytokines, which resulted in death.

A mouse CD8 T cell-mediated acute autoimmune diabetes independent of the perforin and Fas cytotoxic pathways: possible role of membrane TNF.

Double transgenic mice [rat insulin promoter (RIP)-tumor necrosis factor (TNF) and RIP-CD80] whose pancreatic beta cells release TNF and bear CD80 all develop an acute early (6 wk) and lethal diabetes mediated by CD8 T cells. The first ultrastructural changes observed in beta cells, so far unreported, are focal lesions of endoplasmic reticulum swelling at the points of contact with islet-infiltrating lymphoblasts, followed by cytoplasmic, but not nuclear, apoptosis. Such double transgenic mice were made defective in either the perforin, Fas, or TNF pathways. Remarkably, diabetes was found to be totally independent of perforin and Fas. Mice lacking TNF receptor (TNFR) II had no or late diabetes, but only a minority had severe insulitis. Mice lacking the TNF-lymphotoxin (LTalpha) locus (whose sole source of TNF are the beta cells) all had insulitis comparable to that of nondefective mice, but no diabetes or a retarded and milder form, with lesions suggesting different mechanisms of injury. Because both TNFR II and TNF-LTalpha mutations have complex effects on the immune system, these data do not formally incriminate membrane TNF as the major T cell mediator of this acute autoimmune diabetes; nevertheless, in the absence of involvement of the perforin or Fas cytotoxic pathways, membrane TNF appears to be the likeliest candidate.

Identical T cell clones are located within the mouse gut epithelium and lamina propia and circulate in the thoracic duct lymph.

Murine gut intraepithelial (IEL) T cell receptor (TCR)-alpha/beta lymphocytes bearing CD8alpha/13 or CD8alpha/alpha coreceptors have been shown previously to express different oligoclonal TCR beta chain repertoires in the same mouse, in agreement with other evidence indicating that these two populations belong to different ontogenic lineages, with only CD8alpha/beta+ IELs being fully thymus dependent. CD8alpha/beta+, but not CD8alpha/alpha+, T lymphocytes are also present in the lamina propria. Here, we show that CD8alpha/beta+ lymphocytes from the lamina propria and the epithelium are both oligoclonal, and that they share the same TCR-beta clonotypes in the same mouse, as is also the case for CD4alpha T cells. Furthermore, identical T cell clones were detected among CD8alpha/beta IELs and CD8alpha/beta+ blasts circulating into the thoracic duct (TD) lymph of the same mouse, whereas TD small lymphocytes are polyclonal. These findings must be considered in light of previous observations showing that T blasts, but not small T lymphocytes, circulating in the TD lymph have the capacity of homing into the gut epithelium and lamina propria. These combined observations have interesting implications for our understanding of the recirculation of gut thymus-dependent lymphocytes and their precursors, and of the events leading up to the selection of their restricted TCR repertoire.

A role for lymphotoxin beta receptor in host defense against Mycobacterium bovis BCG infection.

To investigate the role of membrane lymphotoxin (LT)alpha1 / beta2 and its LTbeta receptor (LTbetaR) in the protective immune response to Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection, we have used a soluble fusion molecule (LTbetaR-IgG1). LTbetaR-Ig treatment interferes with granuloma formation mainly in the spleen by inhibiting macrophage activation and nitric oxide synthase activity. In addition, a large accumulation of eosinophils was observed in the spleen of LTbetaR-Ig-treated infected mice. Decreased blood levels of IFN-gamma and increased IL-4 were also observed, suggesting that the LTbetaR pathway is important in BCG infection to favor a Th1 type of immune response. The treatment of transgenic mice expressing high blood levels of a soluble TNFR1-IgG3 fusion protein with LTbetaR-Ig resulted in a still higher sensitivity to BCG infection, and extensive necrosis in the spleen. In conclusion, these results suggest that the LTbetaR and the TNFR pathways are not redundant in the course of BCG infection and protective granuloma formation: the LTbetaR pathway appears to be important in spleen granuloma formation, whereas the TNFR pathway has a predominant role in other tissues.

Small bowel enteropathy: role of intraepithelial lymphocytes and of cytokines (IL-12, IFN-gamma, TNF) in the induction of epithelial cell death and renewal.

The small bowel mucosa contains within its villus epithelium a large number of intraepithelial lymphocytes (IEL) which upon activation are cytotoxic and release large quantities of IFN-gamma and TNF; these activities are increased by in vitro exposure to IL-12. Mice injected with IL-12 develop severe damage of the villus epithelial cells, in form of apoptosis, necrosis and a third distinct form of cell death, characterized ultrastructurally by progressive cell shrinkage. These lesions are accompanied by a compensatory acceleration of the epithelial renewal, a hallmark of epithelial injury. Use of a variety of mutant mice showed that these lesions require the presence of IEL (all populations being involved, thymus-dependent as well as natural killer-T cell IEL) and the release of IFN-gamma. The critical role of IFN-gamma may result in part from its capacity to induce on epithelial cells the expression of target molecules involved in the different cytotoxic pathways used by IEL. However, injection of IFN-gamma into mutant mice lacking IEL showed that IFN-gamma can directly induce villus epithelial damage as well. On the other hand, injection of TNF induces fulminant apoptosis of villus epithelial cells, starting at the top of the villi; however TNF is not required for IL-12-induced enteropathy, which is unmodified in mutant mice lacking TNF. We propose that, when activated by their cognate ligands and/or IL-12 produced by cells in the lamina propria, IEL eliminate infected and senescent epithelial cells through a combination of cytotoxicity and of IFN-gamma and TNF release. This insures the rapid epithelial renewal of the villi, which in turn helps maintain the functional integrity of the barrier.

An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.

Transgenic mice expressing high levels of the BclxL or Bcl2 proteins in the male germinal cells show a highly abnormal adult spermatogenesis accompanied by sterility. This appears to result from the prevention of an early and massive wave of apoptosis in the testis, which occurs among germinal cells during the first round of spermatogenesis. In contrast, sporadic apoptosis among spermatogonia, which occurs in normal adult testis, is not prevented in adult transgenic mice. The physiological early apoptotic wave in the testis is coincident, in timing and localization, with a temporary high expression of the apoptosis-promoting protein Bax, which disappears at sexual maturity. The critical role played by the intracellular balance, probably hormonally controlled, of the BclxL and Bax proteins (Bcl2 is apparently not expressed in normal mouse testis) in this early apoptotic wave is shown by the occurrence of a comparable testicular syndrome in mice defective in the bax gene. The apoptotic wave appears necessary for normal mature spermatogenesis to develop, probably because it maintains a critical cell number ratio between some germinal cell stages and Sertoli cells, whose normal functions and differentiation involve an elaborate network of communication.

The role of E-selectin for neutrophil activation and tumor metastasis in vivo.

The selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote metastasis of certain types of tumors, although direct evidence is lacking. To explore this hypothesis two sets of transgenic mice were developed, one TgNES, which constitutively expresses cell surface E-selectin in all tissues, the other TgNEsol, which expresses truncated, soluble E-selectin in the liver. Both transgenic mice showed apparently normal phenotype. However in TgNES mice, but not in TgNEsol mice, the number of neutrophil decreased to 50% compared with that in their negative littermate. And also these neutrophils were markedly activated. On the other hand, B16 F10 melanoma cells were stably transfected with alpha 1-3/4 fucosyltransferase-specific cDNA (B16F10 ft), allowing them to express E-selectin ligands. Normal mice injected with B16F10 ft developed lung tumors exclusively. In contrast, TgNES mice developed massive, infiltrating liver tumors. TgNEsol mice also developed liver tumors that grow more slowly. These observations suggest the important role of E-selectin for neutrophil activation and tumor metastasis in vivo.

Transgenic mice expressing soluble tumor necrosis factor-receptor are protected against bone loss caused by estrogen deficiency.

To evaluate the role of tumor necrosis factor (TNF alpha) in bone loss resulting from estrogen deficiency, the effects of ovariectomy were explored in six-month-old transgenic mice expressing high blood levels of a soluble TNF receptor type I (sTNFR1)-FcIgG3 fusion protein, which neutralizes TNF alpha, and in their nontransgenic littermates used as controls. These transgenic mice were identical to control mice in bone mass (evaluated by bone mineral density and content) and strength. 12 weeks after ovariectomy, the decrease in bone mass and increase in osteocalcin (marker of bone turnover) found in control mice were not observed in transgenic mice, which were not different from sham-operated mice, transgenic or not. This observation suggests a critical role for TNF alpha in the pathogenesis of bone loss induced by estrogen deficiency, a common cause of morbidity in postmenopausal women.

Mouse vaginal opening is an apoptosis-dependent process which can be prevented by the overexpression of Bcl2.

In the mouse, opening of the vaginal cavity to the skin is a late event, occurring around the fifth week of life; it can be induced in sexually immature mice by beta-estradiol injections. We have generated two lines of transgenic mice expressing the human Bcl2 protein in a variety of tissues. The vaginal cavity of the transgenic females remained permanently closed, a condition completely resistant to beta-estradiol injections; this was accompanied by a considerable distension of the genital tract. Histologic studies of vaginal sections at the time of opening to the skin in normal mice showed, by the TUNEL method which detects nuclei with fragmented DNA characteristic of apoptosis, that this event coincides with extensive apoptosis in the lower part of the vaginal mucosa, a process prevented in the bcl2 transgenic mice, which express Bcl2 in suprabasal epithelial cells and in subepithelial cells of the vaginal mucosa. In contrast, two lines of mice bearing a Bcl2 transgene placed under the control of a K10 keratin promoter, whose expression is restricted to the suprabasal layers of the epidermis, had a normal phenotype. Eyelids' formation and opening of the external ear canals, which also occur after birth in the mouse, were not altered in any of these transgenic lines; histological study of eye and ear sections at the time of these events failed to detect apoptosis. In conclusion, the tissue remodeling required to complete maturation of the mouse female genital tract at the time of puberty is an hormonally triggered apoptosis-dependent process.

Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation.

CD44 is a broadly distributed polymorphic glycoprotein that serves as the principal cell-surface receptor for hyaluronate. Although CD44-mediated cell interaction with hyaluronate has been implicated in a variety of physiologic events, including cell-cell and cell-substrate adhesion, cell migration, proliferation, and activation, as well as hyaluronate uptake and degradation, the biologic role of CD44 in vivo in various tissues remains to be determined. In the present work we have developed transgenic mice that express an antisense CD44 cDNA driven by the keratin-5 promoter. These mice lack detectable CD44 expression in skin keratinocytes and corneal epithelium and display abnormal hyaluronate accumulation in the superficial dermis and corneal stroma, distinct morphologic alterations of basal keratinocytes and cornea, and defective keratinocyte proliferation in response to mitogen and growth factors. These alterations are reflected by a decrease in skin elasticity, impaired local inflammatory response and tissue repair, delayed hair regrowth, and failure of the epidermis to undergo hyperplasia in response to carcinogen. Our observations indicate that two major functions of CD44 in skin are the regulation of keratinocyte proliferation in response to extracellular stimuli and the maintenance of local hyaluronate homeostasis.

Evidence for a protective role of tumor necrosis factor in the acute phase of Trypanosoma cruzi infection in mice.

A possible role for tumor necrosis factor (TNF) alpha during Trypanosoma cruzi infection was explored by using transgenic mice expressing in blood high levels of a soluble TNFR1-FcIgG3 fusion protein, which neutralizes the effects of TNF in vivo. Nontransgenic littermates were used as controls. The transgenic mice showed high susceptibility to T. cruzi infection. Inocula sublethal for control mice resulted in over 80% mortality associated with higher levels of parasites in the blood. In histological sections of the hearts of transgenic mice, large parasitic clusters without inflammatory cell infiltrates around the parasites were seen, while smaller parasitic clusters associated with leukocytes were seen in control mice. No difference in specific antibody response or lymphocyte composition of the spleen was found between transgenic and control mice, although the unresponsiveness of spleen cells to concanavalin A stimulation in vitro, typical of the acute phase of T. cruzi infection, was less pronounced in transgenic mice. Infected transgenic mice produced higher levels of gamma interferon than did control mice. These results confirm that TNF is involved in mechanisms leading to parasite clearance and protection from death in the acute phase of T. cruzi infection. More importantly, the data reveal that TNF is necessary for the establishment of effective tissue inflammation and parasite load control in acute experimental Chagas' disease myocarditis.

Prevention of autoimmune diabetes mellitus in NOD mice by transgenic expression of soluble tumor necrosis factor receptor p55.

The non-obese diabetic (NOD) mouse represents a relevant animal model of autoimmunity for insulin-dependent diabetes mellitus. The pathogenic role of tumor necrosis factor (TNF) in insulitis and beta cell destruction observed in these mice remains controversial, since injections of TNF or of anti-TNF antibodies have been reported to exert protection or acceleration of diabetes, depending on the timing of administration. In this study, we demonstrate that, in contrast to the non-transgenic littermates, NOD mice with permanent neutralization of TNF by high blood levels of soluble TNF receptor p55-human FcIgG3-fusion molecules resulting from the expression of a transgene are protected from spontaneous diabetes. They are also protected from accelerated forms of disease caused by transfer of NOD spleen cells or cyclophosphamide injections. This protection is associated with a marked decrease in the severity and incidence of insulitis and in the expression of the adhesion molecules MAdCAM-1 and ICAM-1 on the venules of pancreatic islets. These data suggest a central role for TNF-alpha in the mediation of insulitis and of the subsequent destruction of insulin-secreting beta-cells observed in NOD mice. They may be relevant to cell-mediated autoimmune diseases in general, in which treatment with soluble TNF receptors might be beneficial.

High sensitivity of transgenic mice expressing soluble TNFR1 fusion protein to mycobacterial infections: synergistic action of TNF and IFN-gamma in the differentiation of protective granulomas.

To investigate the role of tumor necrosis factor (TNF) in protective immune responses to Mycobacterium tuberculosis and M. bovis Bacillus Calmette Guérin (BCG), we have used transgenic mice unable to use TNF because of the expression of high amounts of a soluble TNF receptor (R) type I (sTNFR1) fusion protein, and studied resistance of these mice to infection by lethality assays, evaluation of bacterial recovery and histologic examination. These mice showed a strongly increased sensitivity to M. tuberculosis and BCG infections, with bacterial overgrowth and marked inhibition of macrophage differentiation within granulomas; after M. tuberculosis infection, this resulted in extensive lesions of caseous necrosis in the lung. To explore the respective roles of TNF and interferon (IFN)-gamma in resistance to BCG and granuloma differentiation, controls and sTNFR1-transgenic mice were compared to IFN-gammaR mutant mice and mice double defective in TNF and IFN-gamma activity (obtained by crossing transgenic and mutant mice). The three groups of deficient mice showed a strongly enhanced susceptibility to BCG infection, with the following decreasing order of sensitivity between groups: TNF + IFN-gamma --> TNF --> IFN-gamma-deficient mice. The hepatic granulomas of IFN-gammaR mutant mice were small and contained eosinophils but few differentiated macrophages; compared to those of sTNFR1-transgenic mice, acid-fast bacilli were less numerous within the macrophages. Granulomas of double-deficient mice were strikingly different by their very large size and cellular content, made up large numbers of polymorphonuclears, eosinophils, and cells undergoing apoptosis, but without detectable differentiated macrophages; acid-fast bacilli were spread in the lesions. These studies show the essential role of both TNF and IFN-gamma in the development, during mycobacterial infections, of protective granulomas containing highly differentiated macrophages capable of destroying ingested bacteria, and emphasize that these two cytokines act synergistically in granuloma formation.

Systemic injection of a tripeptide inhibits the intracellular activation of CPP32-like proteases in vivo and fully protects mice against Fas-mediated fulminant liver destruction and death.

Mice injected with anti-Fas antibody die within a few hours with total liver destruction due to massive apoptosis of hepatocytes. We show that this is preceded and accompanied by the sequential activation of cysteine proteases of the interleukin 1 beta-converting enzyme (ICE) and CPP32 types in the cytosol of the hepatocytes, and that proCPP32 cleavage and enzymatic activity can be prevented by intravenous injections of the tripeptide N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk), an inhibitor of ICE-like proteases. Four Z-VAD.fmk injections at 1-hour intervals abolished all signs of liver damage after anti-Fas antibody injection and resulted in 100% long-range recovery, without residual tissue damage, from a condition otherwise uniformly fatal within < 3 hours. This treatment was effective even when delayed until some liver DNA degradation was already detectable. Injections of the tetrapeptide Ac-YVAD.cmk, more specific for the ICE-like subfamily of cysteine proteases but less cell permeable, also gave protection, but at higher doses and when injections started before that of anti-Fas antibody. These observations afford a way of temporarily modulating a number of apoptotic processes in vivo and may have important therapeutic implications in some human diseases.

Complexity of the mouse gut T cell immune system: identification of two distinct natural killer T cell intraepithelial lineages.

Gut thymo-dependent (CD8 alpha + beta + or CD4+) or -independent (CD8 alpha + beta -) intraepithelial lymphocytes (IEL) mediate cytotoxicity following T cell receptor (TCR)-CD3 signaling, but only TCR gamma delta + and alpha beta + thymo-independent IEL show cytotoxicity of natural killer (NK) and antibody-dependent cell-mediated cytotoxicity types. Moreover, TCR alpha beta + and gamma delta + thymo-independent IEL express NK receptors, and may therefore be referred to as NK-TIEL. NK-TIEL cytotoxicity is mediated through perforin, Fas, or both pathways. In contrast to that of other NK cells, this cytotoxicity is not negatively regulated by signals delivered through the recognition of major histocompatibility complex class I molecules. Thus, gut IEL include T cell subsets with unique specificities and functions, ontogenically distinct from other T cell lineages, which may increase the antigenic repertoire diversity of the immune system participating in the defense of the epithelial barrier.

E-selectin binding promotes neutrophil activation in vivo in E-selectin transgenic mice.

E-selectin is a membrane protein expressed by endothelial cells activated by cytokines released during the inflammatory process; it plays an important role in neutrophil emigration into inflamed tissues. To further explore in vivo the function of E-selectin, we have generated transgenic mouse line expressing E-selectin under the control of a chicken beta-actin promoter. In these mice, the number of blood neutrophils was reduced, without any other obvious phenotype or tissue damage. These neutrophils, however, displayed two significant changes: first, an alteration in the levels of expression of two membrane receptors involved in neutrophil adhesion to endothelial cells, namely a marked increased in the Mac-1 antigen (CD11b/CD18) and a decrease in the Mel-14 antigen (L-selectin); second, an increased oxidative activity when compared to blood neutrophils of non-transgenic mice, as shown by their capacity to oxidize 2',7'-dichlorofluorescein (DCFH) into a fluorescent compound. These observations indicate that the binding of E-selection with neutrophils bearing its ligands promotes neutrophil activation in vivo.

A bcl-2 transgene expressed in hepatocytes protects mice from fulminant liver destruction but not from rapid death induced by anti-Fas antibody injection.

Stimulation of the Fas (APO-1, CD95) receptor, which is present on a variety of cells, usually triggers a process of programmed cell death. Systemic injection of anti-Fas antibody into mice leads to fulminant liver destruction resulting from massive hepatocyte apoptosis, and to rapid death. Hepatocytes bear Fas but do not express Bcl-2, a protein that plays, in a number of conditions, a protective role against apoptosis. We have generated mice whose liver expresses Bcl-2 as the result of bcl-2 transgene placed under the control of the hepatocyte-specific alpha1-anti-trypsin gene promoter, but is otherwise not distinguishable from that of normal mice. These mice display a marked to almost total resistance to liver damage induced by anti-Fas antibody injection. This protective effect of Bcl-2 occurs in the absence of significant variations, in the stimulated livers, in the level of expression of other proteins also involved in resistance or sensitivity to apoptosis, namely Bcl-x, Bax, Bad, Bak, and p53. Mice with protected livers, however, die almost as rapidly as normal mice, which indicates that acute lethality results from stimulation of Fas receptors present on other target organs or cells.

Redirection of tumor metastasis by expression of E-selectin in vivo.

The selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote tumor cell attachment to endothelium, thereby facilitating metastasis of certain types of tumors, although direct evidence for such a role is lacking. To explore this hypothesis, two sets of transgenic mice were developed: TgnES, which constitutively expresses cell surface E-selectin in all tissues, under the control of the beta-actin promoter; and TgnEsol, which expresses truncated, soluble E-selectin in the liver, under the control of the alpha 1 antitrypsin promoter. B16F10 melanoma cells were stably transfected with alpha(1,3/1,4) fucosyltransferase-specific cDNA (B16F10ft), allowing them to express E-selectin ligands or with hygromycin resistance selection vector only B16F10hygro). Normal mice injected with B16F10ft and B16F10hygro and transgenic mice injected with B16F10hygro developed lung tumors exclusively. In contrast, TgnES mice injected with B16F10ft cells developed massive infiltrating liver tumors. B16F10ft cells injected into TgnEsol mice also formed liver tumors, but these grew more slowly, with a well-delineated, noninfiltrating distinct histologic pattern. These observations provide direct evidence that expression of E-selectin can redirect metastasis of tumor cells expressing appropriate ligands in vivo.