Collagen type II-induced arthritis is a CD4(+) T-cell-dependent chronic inflammation in susceptible DBA/1 mice and represents an animal model of human rheumatoid arthritis. We found that development of this condition, and even established disease, are inhibited by an agonistic anti-4-1BB monoclonal antibody. Anti-4-1BB suppressed serum antibodies to collagen type II and CD4(+) T-cell recall responses to collagen type II. Crosslinking of 4-1BB evoked an antigen-specific, active suppression mechanism that differed from the results of blocking the interaction between 4-1BB and its ligand, 4-1BBL. Anti-4-1BB monoclonal antibodies induced massive, antigen-dependent clonal expansion of CD11c(+)CD8(+) T cells and accumulation of indoleamine 2,3-dioxygenase in CD11b(+) monocytes and CD11c(+) dendritic cells. Both anti-interferon-gamma and 1-methyltryptophan, a pharmacological inhibitor of indoleamine 2,3-dioxygenase, reversed the anti-4-1BB effect. We conclude that the suppression of collagen-induced arthritis was caused by an expansion of new CD11c(+)CD8(+) T cells, and that interferon-gamma produced by these cells suppresses antigen-specific CD4(+) T cells through an indoleamine 2,3-dioxygenase-dependent mechanism.

4-1BB (CD137) is a costimulatory molecule involved in the activation and survival of CD4, CD8, and natural killer cells. Although a great deal has been learned as to how 4-1BB-mediated signaling governs the immunity of conventional T cells, the functional role of 4-1BB in the context of CD4(+)CD25(+) regulatory T cell (Tr) activation is largely unknown. Using 4-1BB-intact and -deficient mice, we investigated the effect of the 4-1BB/4-1BB ligand pathway on the suppressive function of Tr cells. Our data indicate that although 4-1BB is expressed on Tr cells, its contribution to their proliferation is minimal. We also showed that signaling through the 4-1BB receptor inhibited the suppressive function of Tr cells in vitro and in vivo. It is interesting that anti-4-1BB-mediated but not anti-GITR-directed inhibition was more potent when Tr cells were preactivated. Collectively, these data indicate that 4-1BB signaling is critical in Tr cell immunity.

Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43°C for 20 min) resulted in inhibition of H 2 O 2 -induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H 2 O 2 -induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homooligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H 2 O 2 -induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.Apoptosis signal-regulating kinase 1 (ASK1) is a widely expressed serine-threonine kinase that was initially discovered as a mitogen-activated protein kinase kinase kinase (MAPKKK) in the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK signaling cascades (12, 13). ASK1 functions in apoptosis induced by diverse stimuli, including tumor necrosis factor alpha (TNF-␣), Fas, and many apoptotic stresses (12,13,30). Overexpression of a dominantnegative mutant of ASK1 thus prevents apoptosis induced by withdrawal of growth factors, DNA-damaging agents, TNF-␣, or agonistic antibodies to Fas (4,13,16,30). Activation of ASK1 is reported to induce apoptotic cell death by triggering mitochondrial events that include the release of cytochrome c from mitochondria and the subsequent activation of caspase 9 and caspase 3 (11).Many cellular stresses that stimulate the stress-activated MAPK pathways can also induce expression of heat shock proteins. Heat shock protein 72 (Hsp72) is the major inducible heat shock protein (35). It plays a role in many cellular activities including protein synthesis, folding, and translocation into organelles as well as the assembly of multiprotein complexes (2,5,24,25,33). Hsp72 contains two conserved domains, an ATP-binding domain (ABD) and a peptide-binding domain (PBD), that are important for its chaperon function (7, 21). Hsp72 also prevents cell death initiated by various apoptotic stresses including heat shock, ceramide, ionizing irradiation, TNF-␣, and ischemia (8,14,20,23,32). Hsp72 suppresses several apoptotic signaling pathways, including caspase cascades and stress-activated MAPK pathways that include the JNK and p38 signaling cascades (3,8,9,15,17,19,22,23,29,31). Furthermore, Hsp72 has been shown to physically interact with and inhibit Apaf-1 and apoptosis-inducing factor, resulting in suppression of caspase-dependent and -independent apoptosis, respectively ...

Although sperm entry into the oocyte-cumulus complex and subsequent sperm penetration through the cumulus matrix to reach the oocyte zona pellucida are essential for mammalian fertilization, the molecular mechanism remains controversial. Previously, we have shown that mouse sperm lacking SPAM1 are capable of penetrating the cumulus matrix despite a delayed dispersal of cumulus cells. We also have identified another sperm hyaluronidase, HYAL5, as a candidate enzyme involved in sperm penetration through the cumulus. In the present study, we produced HYAL5-deficient mice to uncover the functional roles of HYAL5 and SPAM1 in fertilization. The HYAL5-deficient mice were fully fertile and yielded normal litter sizes. In vitro fertilization assays demonstrated that HYAL5-deficient epididymal sperm is functionally normal. We thus conclude that HYAL5 may be dispensable for fertilization. Comparative analysis among wild-type, HYAL5-deficient, and SPAM1-deficient epididymal sperm revealed that only SPAM1 is probably involved in sperm penetration through the cumulus matrix. Notably, the loss of SPAM1 resulted in a remarkably increased accumulation of sperm on the surface or outer edge of the cumulus. These data suggest that SPAM1 may function in sperm entry into the cumulus and sperm penetration through the cumulus matrix.

Although sperm serine protease and proteasome have long been believed to play an important role in the fertilization process, the molecular mechanism is still controversial. In this study, we have produced double-knockout mice lacking two sperm serine proteases, ACR and PRSS21, to uncover the functional role of the trypsinlike activity in fertilization. The double-knockout male mice were subfertile, likely owing to the incompleteness of fertilization in the oviductal ampulla. Despite male subfertility, the mutant epididymal sperm exhibited the inability to undergo acrosomal exocytosis on the zona pellucida (ZP) surface and to traverse the ZP, thus resulting in the failure of fertilization in vitro. The double-knockout epididymal sperm were also defective in penetration through the cumulus matrix to reach the ZP. When epididymal sperm were artificially injected into the uterus of wild-type mice, the 2-cell embryos, which had previously been fertilized by double-knockout sperm, were recovered at a low but significant level. The mutant epididymal sperm were also capable of fertilizing the oocytes in the presence of uterine fluids in vitro. These data demonstrate that the trypsinlike protease activity of ACR and PRSS21 is essential for the process of sperm penetration through the cumulus matrix and ZP in vitro, and suggest that the female reproductive tract partially compensates for the loss of the sperm function. We therefore conclude that the sperm trypsinlike activity is still important but not essential for fertilization in vivo in the mouse.

Anti-4-1BB-mediated anticancer effects were potentiated by depletion of CD4 + cells in B16F10 melanoma-bearing C57BL/6 mice. Anti-4-1BB induced the expansion and differentiation of polyclonal tumor-specific CD8 + T cells into IFN-;-producing CD11c + CD8 + T cells. The CD4 + cell depletion was responsible for facilitating immune cell infiltration into tumor tissues and removing some regulatory barriers such as T regulatory and indoleamine-2,3-dioxygenase (IDO) + dendritic cells. Both monoclonal antibodies (mAb) contributed to the efficient induction of MHC class I molecules on the tumor cells in vivo. The effectors that mediated the anti-4-1BB effect were NKG2D + KLRG1 + CD11c + CD8 + T cells that accumulated preferentially in the tumor tissues. Blocking NKG2D reduced the therapeutic effect by 20% to 26%, which may indicate that NKG2D contributes partially to tumor killing by the differentiated CD8 + T cells. Our results indicate that the combination of the two mAbs, agonistic anti-4-1BB and depleting anti-CD4, results in enhanced production of efficient tumor-killing CTLs, facilitation of their infiltration, and production of a susceptible tumor microenvironment. [Cancer Res 2007;67(18):8891-9]

Proper biogenesis of a sperm-specific organelle, the acrosome, is essential for gamete interaction. An acrosomal matrix protein, ACRBP, is known as a proacrosin-binding protein. In mice, two forms of ACRBP, wild-type ACRBP-W and variant ACRBP-V5, are generated by pre-mRNA alternative splicing of Acrbp. Here, we demonstrate the functional roles of these two ACRBP proteins. ACRBP-null male mice lacking both proteins showed a severely reduced fertility, because of malformation of the acrosome. Notably, ACRBP-null spermatids failed to form a large acrosomal granule, leading to the fragmented structure of the acrosome. The acrosome malformation was rescued by transgenic expression of ACRBP-V5 in ACRBP-null spermatids. Moreover, exogenously expressed ACRBP-W blocked autoactivation of proacrosin in the acrosome. Thus, ACRBP-V5 functions in the formation and configuration of the acrosomal granule during early spermiogenesis. The major function of ACRBP-W is to retain the inactive status of proacrosin in the acrosome until acrosomal exocytosis.

BackgroundThe wnt/β-catenin signaling pathway is known to affect in cancer oncogenesis and progression by interacting with the tumor microenvironment. However, the roles of wnt3a and wnt5a in colorectal cancer (CRC) have not been thoroughly studied. In the present study, we investigated the expression of wnt protein and the concordance rate in primary tumor and metastatic sites in CRC. To determine the relationship of wnt proteins with invasion related protein, we also analyzed the association between wnt protein expression and the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor receptor-2 (VEGFR-2).MethodsTumor tissue was obtained from eighty-three paraffin- embedded blocks which were using resected tissue from both the primary tumor and metastatic sites for each patient. We performed immunohistochemical staining for wnt3a, wnt5a, β-catenin, MMP-9 and VEGFR-2.ResultsWnt3a, wnt5a, β-catenin, and MMP-9 expression was high; the proteins were found in over 50% of the primary tumors, but the prevalence was lower in tissue from metastatic sites. The concordance rates between the primary tumor and metastatic site were 76.2% for wnt5a and 79.4% for wnt3a and β-catenin, but VEGFR-2 was expressed in 67.4% of the metastatic sites even when not found in the primary tumor. Wnt3a expression in primary tumors was significantly associated with lymph node involvement (p = 0.038) and MMP-9 expression in the primary tumor (p = 0.0387), mesenchyme adjacent to tumor (p = 0.022) and metastatic site (p = 0.004). There was no other relationship in the expression of these proteins. Vascular invasion in primary tumor tissue may be a potential prognostic marker for liver metastasis, but no significant association was observed among the wnt protein, MMP-9, and VEGFR-2 for peritoneal seeding. In survival analysis, β-catenin expression was significantly correlated with overall survival (p = 0.05).ConclusionsWnt3a and wnt5a expression had a concordance rate higher than 60% with a high concordance rate between the primary tumor and metastatic site. Wnt3a expression is associated with the expression of MMP-9 in primary tumor tissue adjacent mesenchymal tissue, and at the metastatic site. As a prognostic marker, only β-catenin expression showed significant relation with survival outcome.