Superantígenos y neoplasias linfoides / SUPERANTIGENS AND NEOPLASTIC LYMPHOMAS

Los superantígenos (Sags) son proteínas de origen bacteriano o viral capaces de estimular y causar la muerte de un alto numero de linfocitos T o B. normales. Este hecho se debe a que ­ a diferencia de los antígenos convencionales que son reconocidos por todas las regiones variables del receptor de las células T (TCR) o B (BCR) - sólo interactúan con regiones constantes ubicadas dentro de las regiones variables de estos receptores. Los Sags B se unen a sitios constantes de las regiones variables de las cadenas pesadas o livianas de las inmonuglobulinas de superficie (BCR) y causan la muerte por apoptosis de las células B que los reconocen. Los Sags T se unen a los antígenos mayores de histocompatibilidad de clase II en las células presentadoras de antígenos e interactúan con linfocitos T que expresen una región variable particular de su cadena â. Las células T reactivas mueren después de recibir esta fuerte estimulación. No se conocen intentos de investigar si los Sags son capaces de inducir la muerte por apoptosis de células neoplásicas B o T. En el presente trabajo reportamos que diferentes Sags bacterianos o virales son capaces de inducir la apoptosis de celulas de leucemias/linfomas B y T murinos y humanos. Estudiamos los mecanismos de apoptosis involucrados. En el caso de los linfomas T estudiados encontramos que estaban implicadas tanto la vía extrínseca cuanto la intrínseca e incluso un entrecruzamiento de ambas vías. El tratamiento in vivo de ratones inoculados con diferentes linfomas T fue capaz de incrementar muy significativamente la sobrevida libre de enfermedad. Mientras que el 100% de los ratones no tratados o tratados con un Sag control (no interactuante con el receptor T del linfoma) morían en pocos días, el tratamiento con Sags específcos indujo una sobrevida de entre el 40 al 90% de los mismos. Se investigo la capacidad del Sag B PpL secretado por Finegoldia magna y que interactua con celulas B que expresan la cadena liviana κ para inducir la apoptosis in vivo e in vitro. de celulas de linfomas/leucemias B murinas y humanas κ+. El Sag utilizado indujo la apoptosis en estas celulas utilizando solo la via intrinseca. En resumen, se demostro que los Sags B y T inducen la muerte por apoptosis de cèlulas B y T neoplasicas que los reconocen a traves de su receptor para el antigeno. Se discute la posibilidad del uso de los superantígenos para la implementacion de una nueva herramienta terapeutica que tendria la ventaja de afectar solo un numero discreto de linfocitos sin afectar otros tipos de cèlulas normales.

Superantiges are mostly bacterial or viral proteins that stimulate a large number of normal T or B lymphocytes.. This fact is because - unlike conventional antigens that are recognized by all the variable regions of the receptor of these cells - they only interact with constant regions located within the variable regions of the receptors. B-cell superantigens bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. T cell Superantigens bind to major histocompatibility complex class II molecules in antigen presenting cells and interact with T cells expressing a particular T cell receptor Vß inducing a strong proliferation/deletion response of the superantigen-reactive T cells. No attempts to investigate whether B or T-cell Sags are able to induce the apoptosis of cognate malignant B or T cells were reported. In the present study we show that bacterial and viral-encoded superantigens induce the apoptosis of murine and human cognate lymphoma T cells both in vitro and in vivo. The extrinsic and the intrinsic patway of apoptosis were found to be involved. Besides, a cross-talk between both pathway was also found. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. We also studied the ability of a B-cell superantigen (PpL) secreted by Finegoldia magna, which interacts with κ+ bearing cells, to induce the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. The involvement of the intrinsic but not the extrinsic patway of apoptosis was clearly demonstrated. In summary, herein we show that B and T superantigens are able to induce the apoptosis of cognate B and T malignat cells.The possibility of a therapeutic use of T and B Superantigens in lymphoma/ leukemia B and T cells is discussed. Their possible use would have the advantage of delete only a discrete number of normal lymphocytes without altering other normal cell types.

A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells.

B-cell superantigens (Sags) bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. No attempts to investigate whether B-cell Sags are able to induce the apoptosis of cognate malignant B cells were reported. In the present study we show that protein L (PpL), secreted by Finegoldia magna, a B-cell Sag which interacts with κ+ bearing cells, induces the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. Apoptosis was not altered by caspase-8 inhibitor. No alterations in the levels of Bid, Fas and Fas-L were found suggesting that PpL does not activate the extrinsic pathway of apoptosis. The involvement of the intrinsic pathway was clearly indicated by: i) alterations in mitochondrial membrane potential (ΔΨm) both in murine and human lymphoma cells exposed to PpL; ii) decreased levels of apoptosis in the presence of caspase-9 inhibitor; iii) significant increases of Bim and Bax protein levels and downregulation of Bcl-2; iv) the translocation from the cytoplasm to the mitochondria of Bax and Bim pro-apoptotic proteins and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor and v) the translocation of Bcl-2 protein from the mitochondria to the cytosol and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor. The possibility of a therapeutic use of Sags in lymphoma/leukemia B cell malignancies is discussed.

B-cell lymphopoiesis is regulated by cathepsin L.

Cathepsin L (CTSL) is a ubiquitously expressed lysosomal cysteine peptidase with diverse and highly specific functions. The involvement of CTSL in thymic CD4+ T-cell positive selection has been well documented. Using CTSL(nkt/nkt) mice that lack CTSL activity, we have previously demonstrated that the absence of CTSL activity affects the homeostasis of the T-cell pool by decreasing CD4+ cell thymic production and increasing CD8+ thymocyte production. Herein we investigated the influence of CTSL activity on the homeostasis of peripheral B-cell populations and bone marrow (BM) B-cell maturation. B-cell numbers were increased in lymph nodes (LN), spleen and blood from CTSL (nkt/nkt) mice. Increases in splenic B-cell numbers were restricted to transitional T1 and T2 cells and to the marginal zone (MZ) cell subpopulation. No alterations in the proliferative or apoptosis levels were detected in peripheral B-cell populations from CTSL (nkt/nkt) mice. In the BM, the percentage and the absolute number of pre-pro-B, pro-B, pre-B, immature and mature B cells were not altered. However, in vitro and in vivo experiments showed that BM B-cell production was markedly increased in CTSL (nkt/nkt) mice. Besides, BM B-cell emigration to the spleen was increased in CTSL (nkt/nkt) mice. Colony-forming unit pre-B (CFU pre-B) assays in the presence of BM stromal cells (SC) and reciprocal BM chimeras revealed that both BM B-cell precursors and SC would contribute to sustain the increased B-cell hematopoiesis in CTSL (nkt/nkt) mice. Overall, our data clearly demonstrate that CTSL negatively regulates BM B-cell production and output therefore influencing the homeostasis of peripheral B cells.

A polymeric protein induces specific cytotoxicity in a TLR4 dependent manner in the absence of adjuvants.

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It is possible to insert foreign peptides or proteins at its ten-amino acid termini. These chimeras elicit systemic and oral immunity without adjuvants, which are commonly needed in the formulation of subunit-based vaccines. Here, we show that BLS induces the cross presentation of a covalently attached peptide OVA(257-264) and a specific cytotoxic response to this peptide in the absence of adjuvants. Unlike other subunit-based vaccines, this chimera induces rapid activation of CTLs and a specific cytotoxic response, making this polymeric protein an ideal antigen carrier for vaccine development. Adoptive transfer of transgenic OT-I T cells revealed efficient cross presentation of BLS-OVA(257-264)in vivo. BLS-OVA(257-264) immunization induced the proliferation of OVA(257-264)-specific CD8+ lymphocytes and also increased the percentage of OVA(257-264)-specific CD8+ cells expressing the early activation marker CD69; after 5 days, the percentage of OVA(257-264)-specific CD8+ cells expressing high levels of CD44 increased. This cell subpopulation showed decreased expression of IL-7Rα, indicating that BLS-OVA(257-264) induced the generation of CD8+ effector cells. BLS-OVA(257-264) was cross presented in vitro independently of the presence of a functional TLR4 in the DCs. Finally, we show that immunization of wild type mice with the chimera BLS-OVA(257-264) without adjuvants induced a strong OVA(257-264)-specific effector cytotoxic response. This cytotoxicity is dependent on TLR4 as is not induced in mice lacking a functional receptor. These data show that TLR4 signaling is necessary for the induction of a cytotoxic response but not for antigen cross presentation.

[Increase of regulatory T cells in the lymph node of cathepsin L mutant mice]. / Aumento de linfocitos T regulatorios en los ganglios linfoides de ratones mutantes para catepsina L.

Regulatory CD4+CD25+Foxp3+ T cells (Treg) have been implicated in different pathologies including cancer, infections and autoimmune diseases and in the rejection of allogeneic organ transplantation. Thus, modulation of Treg activity has a great potential in the treatment of these pathologies. Herein, we evaluated the influence of cathepsin L (CTSL) on Treg homeostasis. CTSL mutant mice (CTSLnkt/nkt) showed a decrease in the absolute number of thymic Treg cells. In contrast, the absolute number of lymph node Treg cells and their frequency within CD4+ cells were increased. The absence of CTSL activity in CD4+ T cells -and not in their environment- increased the proliferation rate of lymph node CD4+ T cells. Treg and T CD4+ conventional (CD4+CD25-Foxp3-) cells from mutant mice showed similar increases in their proliferative levels as compared with control mice, suggesting that although proliferation contributes to the increases in their number, the augmentation in the frequency of Treg cells is not only associated to increases in proliferation. Furthermore, the Treg apoptosis rate was not decreased in the lymph node of CTSLnkt/nkt mice. Taking into account that the daily CD4+ thymic production is diminished in mutant mice, our results suggest that peripheral Treg increases are probably not the result of increased thymic output and raise the possibility that a conversion to Treg phenotype would be favored in the CD4+ T cells peripheral pool of CTSL mutant mice.

[Regulatory T cell depletion increases the number of CD8 cells during mouse mammary tumor virus infection]. / La depleción de las células T regulatorias aumenta el número de las células CD8 durante la infección con el virus del tumor mamario murino.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. We have shown in a natural model of MMTV infection that the virus causes early and progressive increases in superantigen (Sag)-specific CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in Peyer's patches. Herein, we evaluated whether the depletion of Treg cells affects the CD8+ population during milk-borne MMTV infection. At day 6 of infection, the depletion of Treg cells increased the percentage and absolute number of CD8+ cells in lymph nodes as well as the mean intensity fluorescence of the CD44 activation marker. The absolute number of CD8+ cells was increased in cells bearing both Sag reactive and non-reactive TCR Vß chains. We have previously shown that regulatory T cell depletion at day 6 of infection decrease viral load. Results reported herein suggest that at least after day 6 of MMTV infection Treg cells play an inhibiting role on CD8 antiviral response.

Increases in IgA(+) B cells in Peyer's patches during milk-borne mouse mammary tumor virus infection are influenced by Toll-like receptor 4 and are completely dependent on the superantigen response.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Although mammary glands are the final target of infection, Peyer's patches (PP) are the entry site of the virus. Herein, we show that the infection induces increases in the number of PP IgA(+) B cells and higher expression of the α circular transcript, which is a specific marker of the switch to IgA. In addition, IgA(+) B-cell increases correlated with higher levels of cytokines related to IgA class switching, such as interleukin (IL)-5 and IL-6. Of interest, the increases in IgA(+) B cells were lower in Toll-like receptor 4-deficient mice and were completely dependent on the presence of superantigen-reactive T cells. Our results point to a novel mechanism involved in MMTV infection and suggest that IgA(+) B cells may play an important role in carrying the virus to the mammary glands.

Central role of extracellular signal-regulated kinase and Toll-like receptor 4 in IL-10 production in regulatory dendritic cells induced by Trypanosoma cruzi.

Several Trypanosoma cruzi molecules that stimulate macrophages activity were described as Toll-like receptor 2 (TLR2) ligands. Besides, the models of dendritic cells (DC) are poorly characterised. We have previously demonstrated that live-trypomastigotes (Tp) plus lipopolysaccharide (LPS) induce DC with tolerogenic properties that produce high levels of interleukin (IL)-10 and an impaired capacity to induce lymphoproliferation. Here, we show that the regulatory phenotype was observed with heat-killed trypomastigotes (Tphk) stimulation, ruling out DC infection. T. cruzi induced a particular DC activation state increasing LPS-activation of extracellular regulated kinase (ERK) 1/2 and signal transducer and activator of transcription (STAT) 3. Inhibition of ERK down-regulated IL-10 production and restored DC stimulatory capacity, showing the importance of this pathway in the DC modulation. A recent work shows that signalling via TLR4 and TLR2 induces a synergism in anti-inflammatory cytokine production in murine DC. Upon TLR2 and TLR4 stimulation using Pam(3)Cys or LPS and Tphk in DC from TLR2 knock out (KO) or TLR4-mutant mice, we showed that high levels of IL-10 were independent of TLR2 but associated with TLR4 and NF-kappaB signallization. Although sialic acid has been described as a molecule responsible of DC inhibition, we determine that it is not associated with T. cruzi-IL-10 modulatory response. In conclusion, all these findings demonstrate a key role of ERK and TLR4 in association with NF-kappaB in IL-10 modulation induced by T. cruzi and suggest that this regulatory effect involves parasite-DC interactions not described yet.

Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.

In recent years it has become clear that the therapeutic properties of bone marrow-derived mesenchymal stromal cells (MSC) are related not only to their ability to differentiate into different lineages but also to their capacity to suppress the immune response. We here studied the influence of MSC on macrophage function. Using mouse thioglycolate-elicited peritoneal macrophages (M) stimulated with LPS, we found that MSC markedly suppressed the production of the inflammatory cytokines TNF-alpha, IL-6, IL-12p70 and interferon-gamma while increased the production of IL-10 and IL-12p40. Similar results were observed using supernatants from MSC suggesting that factor(s) constitutively released by MSC are involved. Supporting a role for PGE(2) we observed that acetylsalicylic acid impaired the ability of MSC to inhibit the production of inflammatory cytokines and to stimulate the production of IL-10 by LPS-stimulated M. Moreover, we found that MSC constitutively produce PGE2 at levels able to inhibit the production of TNF-alpha and IL-6 by activated M. MSC also inhibited the up-regulation of CD86 and MHC class II in LPS-stimulated M impairing their ability to activate antigen-specific T CD4+ cells. On the other hand, they stimulated the uptake of apoptotic thymocytes by M. Of note, MSC turned M into cells highly susceptible to infection with the parasite Trypanosoma cruzi increasing more than 5-fold the rate of M infection. Using a model of inflammation triggered by s.c. implantation of glass cylinders, we found that MSC stimulated the recruitment of macrophages which showed a low expression of CD86 and the MHC class II molecule Ia(b) and a high ability to produce IL-10 and IL-12p40, but not IL-12 p70. In summary, our results suggest that MSC switch M into a regulatory profile characterized by a low ability to produce inflammatory cytokines, a high ability to phagocyte apoptotic cells, and a marked increase in their susceptibility to infection by intracellular pathogens.

Superantigens increase the survival of mice bearing T cell lymphomas by inducing apoptosis of neoplastic cells.

Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vß inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vß region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.

Early increases in superantigen-specific Foxp3+ regulatory T cells during mouse mammary tumor virus infection.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Here, we show in a natural model of MMTV infection that the virus causes early and progressive increases in superantigen (SAg)-specific Foxp3(+) regulatory T cells (T(reg)) in Peyer's patches (PP). These increases were shown to be dependent on the presence of dendritic cells. CD4(+) CD25(+) T cells from the PP of infected mice preferentially suppress the proliferative response of T cells to SAg-expressing antigen-presenting cells ex vivo. We investigated the influence of the depletion of CD25(+) cells at different stages of the infection. When CD25(+) cells were depleted before MMTV infection, an increase in the number of PP SAg-cognate Foxp3(-) T cells was found at day 6 of infection. Since the SAg response is associated with viral amplification, the possibility exists that T(reg) cells attenuate the increase in viral load at the beginning of the infection. In contrast, depletion of CD25(+) cells once the initial SAg response has developed caused a lower viral load, suggesting that at later stages T(reg) cells may favor viral persistence. Thus, our results indicated that T(reg) cells play an important and complex role during MMTV infection.

Critical role of dendritic cells in mouse mammary tumor virus in vivo infection.

Mouse mammary tumor virus (MMTV) is a milk-transmitted betaretrovirus that causes mammary tumors in mice. Although mammary epithelial cells are the ultimate targets of MMTV, the virus utilizes components of the host immune system to establish infection. Previous studies indicated that dendritic cells play a role in MMTV infection. Here we show that dendritic cells are the first cells to be infected by MMTV in vivo and that they are capable of producing infectious virus that can be transmitted to other cell types. Moreover, upon contact with the virus, dendritic cells became more mature and migrated in response to the chemokine macrophage inflammatory protein 3beta. Finally, we demonstrate that targeted ablation of dendritic cells in vivo dramatically attenuated MMTV infection. These data indicate that MMTV infection of dendritic cells is critical to initial propagation of the virus in vivo.

Endogenous glucocorticoids cause thymus atrophy but are protective during acute Trypanosoma cruzi infection.

The cytokine-mediated stimulation of the hypothalamus-pituitary-adrenal (HPA) axis is relevant for survival during bacterial endotoxemia and certain viral infections. However, only limited information is available regarding the effects of endogenous glucocorticoids on parasite diseases. We have studied this issue using, as a model, C57Bl/6 and Balb/c mice infected with Trypanosoma cruzi, the causal agent of Chagas' disease. These two mouse strains differ in the susceptibility to infection with the parasite. An intense stimulation of the HPA-axis was observed 3 weeks after infection in both strains, but glucocorticoid levels were already increased two- to threefold in the less susceptible Balb/c strain during the first week. Blockade of glucocorticoid receptors with the glucocorticoid antagonist RU486, starting on day 10 after infection, partially reversed the thymic atrophy and decreased the number of CD4(+)CD8(+) thymocytes without affecting parasitemia and the number of inflammatory foci in the heart. However, tumor necrosis factor-alpha blood levels were increased in infected mice of both strains treated with RU486. Furthermore, the blockade of glucocorticoid receptors accelerated death in C57Bl/6J mice and increased lethality to 100% in Balb/c mice. The results obtained represent the first evidence that an endocrine host response that is coupled to the immune process can strongly affect the course of a parasite infection.

A polymeric bacterial protein activates dendritic cells via TLR4.

The enzyme lumazine synthase from Brucella spp. (BLS) is a highly immunogenic protein that folds as a stable dimer of pentamers. It is possible to insert foreign peptides and proteins at the 10 N terminus of BLS without disrupting its general folding, and these chimeras are very efficient to elicit systemic and oral immunity without adjuvants. In this study, we show that BLS stimulates bone marrow dendritic cells from mice in vitro to up-regulate the levels of costimulatory molecules (CD40, CD80, and CD86) and major histocompatibility class II Ag. Furthermore, the mRNA levels of several chemokines are increased, and proinflammatory cytokine secretion is induced upon exposure to BLS. In vivo, BLS increases the number of dendritic cells and their expression of CD62L in the draining lymph node. All of the observed effects are dependent on TLR4, and clearly independent of LPS contamination. The described characteristics of BLS make this protein an excellent candidate for vaccine development.

Binding of natural variants of staphylococcal superantigens SEG and SEI to TCR and MHC class II molecule.

SEG and SEI are staphylococcal superantigens (SAgs) identified recently that belong to the egc operon and whose genes are in tandem orientation. Only a few allelic variants of SEG and SEI have been reported. Here we analyzed four Staphylococcus aureus strains with genotypic variation in both SAgs. However, both SAgs retain key residues in their putative TCR and MHC binding sites and, accordingly, their superantigenic properties. Thus, SEI significantly stimulates mouse T-cells bearing Vbeta3, 5 and 13, while SEG stimulates Vbeta7 and 9 in the draining node when inoculated in the footpad. As another member of the SEB subfamily, SEG also stimulates mouse Vbeta8.1+2. However, the increase in Vbeta8.1+2 T-cells observed at day 2 after inoculation reverts to normal values at day 4, whereas it remains high at day 4 following inoculation with SEC3 or SSA. T-cell stimulation assays in the mouse and analysis of the putative Vbeta8.2 binding site on SEG, which includes three non-conserved residues, suggest a possibly unique interaction between Vbeta8.2 and SEG. We also analyzed biochemical and biophysical characteristics of SEI and SEG binding to their cognate human beta chains by surface plasmon resonance, and binding to the HLA-DR1 MHC class II molecule by gel filtration. SEI binds human Vbeta5.2 and Vbeta1 with apparent K(D)'s of 23 and 118 microM, respectively; SEG binds Vbeta13.6 with a K(D) of 5 microM. As suggested by sequence homology, SEI requires Zn2+ for strong binding to DR1, which goes undetected in the presence of EDTA. SEG and SEI have characteristics such as co-expression, different interaction with MHC class II and stimulation of completely different subsets of human and mouse T-cells, which indicate complementary superantigenic activity and suggest an important advantage to staphylococcal strains in producing them both.

Cathepsin-L influences the expression of extracellular matrix in lymphoid organs and plays a role in the regulation of thymic output and of peripheral T cell number.

Nackt mice, which are deficient in cathepsin-L (CTSL), show an early impairment during positive selection in the context of class II MHC molecules and as a consequence, the percentage and absolute number of CD4(+) thymocytes are significantly decreased. In this study, we show that lymph nodes from nackt mice are hypertrophied, showing normal absolute numbers of CD4(+) T cells and marked increases in the number of CD8(+) T lymphocytes. Basal proliferative levels are increased in the CD4(+) but not in the CD8(+) population. Lymph node T cells show increases in the expression of alpha(5), alpha(6), and beta(1) integrin chains. These alterations correlate with increases in the expression of extracellular matrix (ECM) components in lymph nodes. Interestingly, laminin, fibronectin, and collagen I and IV are markedly decreased in nackt thymus which shows an augmented output of CD8(+) cells. These results demonstrate that a mutation in the Ctsl gene influences the levels of ECM components in lymphoid organs, the thymic output, and the number of T cells in the periphery. They further raise the possibility that, by regulating the level of expression of ECM components in lymphoid organs, CTSL is able to broadly affect the immune system.

Thymocyte depletion during acute Trypanosoma cruzi infection in C57BL/6 mice is partly reverted by lipopolysaccharide pretreatment.

Infection with Trypanosoma cruzi in C57BL/6 mice leads to a progressive fatal disease accompanied by thymocyte depletion, which is not related with a higher parasite burden but with increased serum levels of tumour necrosis factor alpha (TNF- alpha). Because this situation may result from an excessive inflammatory syndrome, mice were now given anti-TNF-alpha mAbs throughout their acute infection, or subjected to a LPS desensitization protocol before parasite challenge. Treatment with anti-TNF-alpha mAbs failed to ameliorate thymocyte depletion but shortened survival time and increased parasite load. Pretreatment with LPS (desensitization followed by a sublethal LPS dose) prolonged survival time with a trend to reduce parasitemias and TNF-alpha serum concentrations. Given that pentoxifylline (PTx) interferes with in vitro LPS tolerance, experiments by administering PTx in combination with the tolerance-inducing LPS doses were also performed. Such schedule significantly reduced mortality, TNF-alpha and IL-6 serum concentrations, and CD4+ CD8+ thymocyte loss. LPS pretreatment allowed a better infection control and protected from the accompanying tissue damage.

Toll-like receptor 4-dependent activation of dendritic cells by a retrovirus.

Mouse mammary tumor virus (MMTV) is a milk-borne retrovirus that exploits the adaptive immune system. It has recently been shown that MMTV activates B cells via Toll-like receptor 4 (TLR4), a molecule involved in innate immune responses. Here, we show that direct virus binding to TLR4 induced maturation of bone marrow-derived dendritic cells and up-regulated expression of the MMTV entry receptor (CD71) on these cells. In vivo, MMTV increased the number of dendritic cells in neonatal Peyer's patches and their expression of CD71; both these effects were dependent on TLR4. Thus, retroviral signaling through TLRs plays a critical role in dendritic-cell participation during infection.

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution.

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.