TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development.

Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-beta (TGF-beta), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-beta inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-beta responses, any role of TGF-beta in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-beta promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant-negative mutant (TbetaRIIDeltacyt) of the TGF-beta type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-beta. In a murine model of bone metastases, expression of TbetaRIIDeltacyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant-negative signaling blockade by expression of a constitutively active TGF-beta type I receptor in the breast cancer cells increased tumor production of parathyroid hormone-related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant-negative TbetaRIIDeltacyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-beta in the development of breast cancer metastasis to bone, via the TGF-beta receptor-mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.

A role for interleukin-6 in host defense against murine Chlamydia trachomatis infection.

Interleukin-6-deficient (IL-6(-/-)) knockout mice had significantly increased Chlamydia trachomatis levels in lung tissue and increased mortality compared to B6129F2/J controls early after intranasal infection. Gamma interferon production and chlamydia-specific antibody levels were consistent with a decreased but reversible Th1-like response in IL-6(-/-) mice. IL-6 is needed for an optimal early host response to this infection.

Humoral and cellular immunity in secondary infection due to murine Chlamydia trachomatis.

A murine model of pneumonia due to the mouse pneumonitis agent (MoPn [murine Chlamydia trachomatis]) in mice deficient in CD4+ T-cell function (major histocompatibility complex [MHC] class II function [class II-/-], CD8+ T-cell function (beta2-microglobulin deficient, MHC class I deficient [Beta2m-/-]), B-cell function (C57BL/10J-Igh(tm1Cgn) [Igh-/-]), and gamma interferon (IFN-gamma) (C57BL/6-Ifg(tm1) [Ifg-/-]) or interleukin-4 (C57BL/6J(tm1Cgn29) [IL4-/-]) production was employed to determine if each of these mechanisms was critical to resistance against reinfection by C. trachomatis or if alternate compensatory mechanisms existed in their absence which could potentially be exploited in vaccine development. Resistance to reinfection with MoPn was heavily dependent on CD4+ T cells. CD4 T-cell-deficient MHC class II-/- mice were very susceptible to reinfection with MoPn, showing the critical importance of this cell to resistance. These mice lacked antibody production but did produce IFN-gamma, apparently by mechanisms involving NK and CD8+ T cells. Neutralization of IFN-gamma in these mice led to a borderline increase in susceptibility, showing a possible role (albeit small) of this cytokine in this setting. Tumor necrosis factor alpha (TNF-alpha) was also present at increased levels in these mice. Igh-/- B-cell-deficient mice which produce no antibody to MoPn were only modestly more susceptible to reinfection than immunized B-cell-intact controls, showing that antibody, including lung immunoglobulin A, is not an absolute requirement for relatively successful host defense in this setting. Levels of lung IFN-gamma and TNF-alpha were elevated in Igh-/- mice compared to those in controls. IL-4-/- mice (deficient in Th2 function) could develop normal resistance to reinfection with MoPn. Conversely, normal mice rendered partially IFN-gamma deficient by antibody depletion were somewhat impaired in their ability to develop acquired immunity to MoPn, again indicating a role for this cytokine in host defense against rechallenge. Of most importance, however, congenitally IFN-gamma-deficient Ifg-/- mice (which have elevated levels of other cytokines, including TNF-alpha and granulocyte-macrophage colony-stimulating factor) are paradoxically more resistant to MoPn rechallenge than controls, showing that IFN-gamma is not an absolute requirement for acquired resistance and implying the presence of very effective compensatory host defense mechanism(s). In vivo depletion of TNF-alpha significantly increased MoPn levels in the lungs in these mice. Thus, resistance to reinfection in this model is flexible and multifactorial and is heavily dependent on CD4+ T cells, with a probable role for IFN-gamma and TNF-alpha and a possible modest role for Th1-dependent antibody. Since IFN-gamma was dispensable in host defense, the highly effective mechanism or mechanisms which can compensate for its absence (which include TNF-alpha) deserve further study.

Role of gamma-delta T cells in murine Chlamydia trachomatis infection.

The role of gamma-delta T cells in host resistance to Chlamydia trachomatis was characterized by using a murine model of pneumonia caused by the mouse pneumonitis agent (MoPn), murine C. trachomatis. At days 3 and 7 after infection, gamma-delta T-cell-deficient knockout mice had significantly higher levels of MoPn in the lungs than did immunologically intact controls. At day 20, paradoxically, gamma-delta T-cell-deficient mice were more resistant to MoPn than were controls. This increased resistance was not due to an increased production of toxic cytokines or interleukin-10 in controls on that day. Gamma-delta T cells play a role in protection early in MoPn infection, but they may be deleterious later in infection, as has been observed in models of salmonella and trypanosome infection.

Activation of latent transforming growth factor beta during Chlamydia trachomatis-induced murine pneumonia.

Transforming growth factor beta (TGF beta) is a multifunctional cytokine with potentially important roles in both host defence and immunopathogenesis. Latent, but more importantly, active TGF beta was significantly elevated in bronchiolar lavage fluid from lungs of mice infected with murine Chlamydia trachomatis. Induction of both latent and active TGF beta in these infected animals was highest at day two after infection (2 to 4-fold) compared with day 15 (1 to 2-fold). Both active and latent TGF beta 1 and TGF beta 2 isoforms were detected. Quantitative reverse transcription polymerase chain reaction (RT-PCR) assay showed a slight but significant increase in PCR product for TGF beta 1, but Northern analysis for TGF beta 1 in lung tissue was not significantly different between treatment groups. No significant change was observed for TGF beta 2 mRNA by RT-PCR. The increase in active and latent TGF beta in these lung lavages from mice infected with C. trachomatis appears to be primarily post-transcriptionally regulated.

Role of CD8 T cells in primary Chlamydia infection.

The role of CD4 and CD8 T cells in primary Chlamydia trachomatis pneumonia was investigated by using in vivo depletion techniques to eliminate T-cell populations. Reduction of either CD4 T cells or CD8 T cells caused a significant increase in organism burden in the lungs, as measured by both quantitative culture and detection of chlamydial antigen on day 14 postinfection. Chlamydia-specific antibody levels in plasma or antigen-induced gamma interferon (IFN-gamma) production by spleen cells was dramatically reduced by depletion of CD4 cells. The reduction in IFN-gamma achieved by depletion of CD8 cells did not reach statistical significance. In the survival studies, depletion of CD4 cells led to a significant increase in mortality. Although there was a trend toward higher mortality, depletion of CD8 cells did not significantly increase mortality. The role of CD8 T cells in host defense was clarified in studies using beta 2-microglobulin-deficient (major histocompatibility class I antigen-deficient, C1D) mice which are defective in CD8 T-cell function. In this model, a significant increase in organism burden was seen during infection in C1D mice compared with that C57BL/6 controls and a significant increase in mortality was observed as well. However, surviving C1D mice were able to clear the infection by day 34. C1D mice had increased numbers of CD4 T cells in both the spleen and the lungs during infection compared with those of C57BL/6 controls. IFN-gamma in C57BL/6 mice was produced by both CD4 and CD8 cells. Thus, there is a protective role for both CD4 and CD8 cells in host defense against Chlamydia infection, but the former appear to be dominant.

Alternative routes of immunization for the induction of experimental autoimmune uveoretinitis (EAU) in rodents: a comparison.

Experimental autoimmune uveoretinitis (EAU) in rodents is a widely used model of ocular autoimmunity. EAU has traditionally been elicited by injecting the uveitogenic protein in complete Freund's adjuvant (CFA) into the footpad(s) (FP). Because this route of immunization causes severe arthritis and inflammation, it is being banned by many institutions and investigators are switching to the subcutaneous (SC) route. However, there are no studies that systematically compare the outcome of these two immunization routes using defined clinical, histopathological and immunological criteria. We therefore undertook to compare the FP and SC routes of immunization in the Lewis rat and in the B 10. A mouse models of EAU. Animals were immunized with interphotoreceptor retinoid-binding protein (IRBP) or the retinal soluble antigen (S-Ag) in CFA, either by the traditional FP route or by the SC route. The parameters studied were kinetics and severity of EAU by clinical observation and by histopathology, respectively, as well as immunological responses by delayed-type hypersensitivity (DTH), serum antibody titers and lymphocyte proliferation to the uveitogen. In mice immunized with graded doses of IRBP, development of disease induced by the FP and SC methods had essentially identical kinetics. However, the SC method resulted in a somewhat higher incidence and severity of disease as well as higher DTH at the lower antigen doses. Antibody titers tended to be higher with FP immunization. In rats immunized with S-Ag, kinetics and severity of disease, DTH, proliferative responses of draining lymph node cells to the immunizing antigen, and serum antibody titers induced by FP and SC methods were similar. In rats immunized with IRBP, SC immunization resulted in somewhat higher responses across the board than FP. We conclude that at higher doses of antigen disease scores and immunological responses in animals immunized SC are comparable to those of FP-immunized animals. At limiting doses of antigen, however, the SC route appears to result in more severe disease than the traditional FP method.

Endogenous systemic IFN-gamma has a protective role against ocular autoimmunity in mice.

Locally produced IFN-gamma has been implicated in enhancing inflammation and in promoting organ-specific autoimmunity. In the present study, we investigated the influence of systemically available IFN-gamma on the expression of experimental autoimmune uveoretinitis (EAU) induced in mice with the retinal Ag, interphotoreceptor retinoid binding protein (IRBP). EAU-susceptible B10.A mice treated with a mAb to IFN-gamma developed much more severe EAU than did the controls and had increased delayed hypersensitivity (DH) responses to IRBP. There was an increase in the proportion of macrophage/monocytes vs lymphocytes in the ocular lesions of treated animals. The anti-IFN-gamma treatment did not prevent expression of MHC class II within the ocular tissues. Conversely, treatment with rIFN-gamma ameliorated EAU expression and lowered DH responses. This occurred despite widespread induction of MHC class II Ag in the ocular tissues and other organs. In contrast to EAU and DH, serum antibody titers and lymphocyte proliferation to IRBP were not significantly affected by either treatment. Experiments in several genetically resistant strains of mice showed that treatment with anti-IFN-gamma was able to up-regulate disease expression also in some EAU-resistant strains. In the case of one such strain, resistance to EAU induction was completely abrogated by the treatment. We conclude that endogenously produced IFN-gamma at the systemic level acts to down-regulate EAU in the mouse and that IFN-gamma-related mechanisms may be involved in conferring resistance to EAU in some mouse genotypes.

Chlamydia trachomatis pneumonia in the severe combined immunodeficiency (SCID) mouse.

We have developed a model of pneumonia caused by the mouse pneumonitis agent (MoPn, murine Chlamydia trachomatis) in the C.B-17 severe combined immunodeficiency (SCID) mouse. In contrast to our prior models in the nude athymic (nu/nu) and heterozygous (nu/+) mouse, SCID mice lack B-cell function and gamma delta T-cell function. SCID mice were more susceptible to MoPn than nu/nu or nu/+ mice both by criteria of mortality and quantitative lung culture. SCID mice could be reconstituted with thymocytes to be more resistant to MoPn (in the absence of significant antibody production), but the protection was modest and less than that in T-cell reconstituted nu/nu mice in our previous studies. A nu/+ MoPn-specific T-cell clone with a Th1-like cytokine profile also provided modest but significant protection without significant antibody production. The SCID mouse is a useful model to study T-cell-mediated immunity to MoPn in a B cell and gamma delta T-cell-deficient environment.

Gamma interferon levels during Chlamydia trachomatis pneumonia in mice.

Host defense against murine Chlamydia trachomatis (mouse pneumonitis agent [MoPn]) in a murine model was investigated. Gamma interferon (IFN-gamma) was produced in the lungs by both MoPn-susceptible nude athymic (nu/nu) and MoPn-resistant heterozygous (nu/+) mice. In vivo depletion of IFN-gamma in nu/nu mice led to exacerbation of infection. Fluorescence-activated cell sorter analysis disclosed induction of GL3 antibody-positive cells (putatively gamma/delta+ T cells) in nu/nu mouse lung during infection with MoPn. Treatment of nu/nu mice in vivo with antibody to NK cells (anti-asialo GM1 antibody) or to gamma/delta cells (UC7-13D5) did not significantly decrease IFN-gamma production in the lung. However, treatment of severe combined immunodeficiency mice (which lack gamma/delta cells) with antibody to NK cells significantly reduced lung IFN-gamma levels.

Genetic control of susceptibility to experimental autoimmune uveoretinitis in the mouse model. Concomitant regulation by MHC and non-MHC genes.

Experimental autoimmune uveoretinitis (EAU) in animals is a T cell-mediated autoimmune response directed against cells of the neural retina, in particular the photoreceptors. EAU can be induced in susceptible strains of mice by immunization with purified retinal Ag, and serves as a model for human uveitis. Because strong HLA associations have been noted in a number of human uveitic diseases, we investigated the role of MHC vs non-MHC genes in the control of susceptibility to ocular autoimmunity using the mouse EAU model. Selected strains representing most of the known independent H-2 haplotypes, as well as several H-2-recombinant and congenic strains, were immunized with interphotoreceptor retinoid-binding protein. Ocular pathology was induced in strains of the H-2k haplotype and their I-A-matched congenics, as well as in strains of the H-2r, H-2b, and H-2d haplotypes. In a series of experiments utilizing intra-H-2 recombinant strains, MHC control of susceptibility was tentatively mapped to the I-A subregion of the H-2k. Expression of the I-Ek gene product was not required for susceptibility to EAU, and in fact appeared to have an ameliorating effect on disease. Incidence and severity of disease obtained in strains sharing the same H-2 on a different background, or sharing the same background in the context of a different H-2, indicated that non-MHC genes contribute significantly to the regulation of EAU. Disease expression of susceptible H-2 haplotypes was highest in strains with B10 background (permissive) and ranged from intermediate to absent in strains with other (nonpermissive) backgrounds. The data suggest that although the ability to develop ocular pathology is dependent on the I-A subregion of the H-2, the final expression of disease in susceptible haplotypes is largely determined by background, non-MHC genes.

Chlamydia trachomatis pneumonia in the immune, athymic and normal BALB mouse.

This paper compares the histopathology of pneumonia due to murine Chlamydia trachomatis (MoPn, mouse pneumonitis agent) in susceptible athymic nude mice (nu/nu), resistant heterozygous littermates (nu/+) and very resistant immunized nu/+ mice. While all groups had an early heterophil response, successful host defence correlated with the presence of large numbers of plasma cells, lymphocytes, monocytes, and lipid laden macrophages. Reticulate bodies were seen in all groups, predominantly in type I alveolar epithelial cells. By 24 h in the immune nu/+ group, no intact organisms were visible. Optimal control of infection was thus rapid and not clearly related to heterophils. These studies show that the histopathology of chlamydial infection may be quite atypical in the immunocompromised host, mononuclear cells seem critical in host defence, and B cell activation with plasma cell infiltration is dependent on intact T cell function in this model.

Effects of 1,2-dimethylhydrazine treatment and feeding regimen on rat colonic epithelial cell proliferation.

The present study was designed to test the hypothesis that 1,2-dimethylhydrazine dihydrochloride (DMH) induces preneoplasia in rat colonic epithelium and that this DMH-altered epithelium will respond differently to various nutritional challenges in comparison to normal colonic epithelium. Preneoplasia was arbitrarily defined as an altered and irreversible state of colonic epithelial cell proliferation induced by a carcinogen (DMH). In summary, DMH was found to be specific for the enhancement of rat colonic epithelial cell proliferation compared to other rapidly renewing cell populations, i.e., ileal epithelium and ear epidermis. DMH-induced changes in rat colonic epithelial cell proliferation and crypt cellularity were found to be irreversible following a 2- to 8-week recovery period. The p.o. administration of the solid and liquid diets, regardless of chemical constituents, supported a DMH-induced increase in colonic epithelial cell proliferation; however, a DMH-induced increase in epithelial cell proliferation was not observed in rats maintained on total parenteral nutrition. Thus, the route of administration has a significant influence on epithelial cell proliferation in colonic epithelium of DMH-treated rats. The importance of these results, along with previous studies, is the establishment and initial characterization of an exploitable preneoplastic system in rat colonic epithelium. Particularly revealing was the finding that significant changes in crypt kinetic parameters induced by DMH treatment did not revert to control values following a 2- to 8-week recovery period. Based on an altered and irreversible state of colonic epithelial cell proliferation induced by DMH, it is concluded that: (a) the preneoplastic state is a committed state and is not dependent upon the continued presence of the carcinogen; and (b) all cryptal epithelium is preneoplastic, although not all cells progress to the overtly transformed state. In addition, total parenteral nutrition prevented the expression of a DMH-induced preneoplastic state of altered epithelial cell proliferation.