Inhibition of apoptosis signal-regulating kinase 1 enhances endochondral bone formation by increasing chondrocyte survival.

Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has clinical potential to treat fractures or to slow osteoarthritic progression by enhancing chondrocyte survival and slowing hypertrophy.

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation.

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.

Docetaxel alone or orally combined with 5-fluorouracil and its derivatives: effects on mouse mammary tumor cell line MM2 in vitro and in vivo.

Although docetaxel (Taxotere; TXT), a taxoid anticancer drug, is clinically and experimentally very effective against breast cancer, its antitumor effect is of very short duration. We addressed whether 5-fluorouracil (5-FU) and its derivatives can act synergistically with TXT against mammary tumors, with placing particular stress on their use by oral route. Mouse mammary tumor cell line, MM2, was propagated in culture and as ascites in mice. Carmofur (HCFU) and doxifluridine (5'-DFUR) were used as 5-FU derivatives. In vitro, the cytotoxic effects of antitumor drugs on MM2 cells were examined by MTS assay. In vivo, mice inoculated i.p. with MM2 cells were treated with i.p. injection of TXT and/or oral administration of 5-FU or its derivatives, and observed for curing tumor. In vitro, the synergistic effects were observed in the combination of TXT and 5-FU or HCFU, but not in that of TXT and 5'-DFUR. In vivo, all of these combinations cured tumors far more effectively than TXT alone. The discrepant result of the combination of TXT and 5'-DFUR between in vitro and in vivo was ascribed to up-regulation of pyrimidine phosphorylase in tumor cells in vivo by TXT. Thus, 5-FU, its masked compounds like HCFU and its prodrugs like 5'-DFUR can act synergistically with TXT in the therapy of cancer even when administered by the oral route.

Clear suppression of Th1 responses but marginal amelioration of autoimmune manifestations by IL-12p40 transgene in MRL-FAS(lprcg)/FAS(lprcg) mice.

To investigate the effects of overproduction of IL-12p40, a potent antagonist against IL-12, on lupus-like autoimmune disease in vivo, we generated p40 transgenic MRL-Fas(lprcg)/Fas(lprcg) mice. Serum p40 and IL-12 levels were 600- to 8000-fold and 3- to 20-fold higher in transgenic (p40-lpr(cg)) than nontransgenic (lpr(cg)) mice, respectively. Serum IFN-gamma levels increased after 3 months of age in lpr(cg) and this age-related increase was completely abrogated in p40-lpr(cg). Serum IL-4 levels were the same in both mice. Production of IgM and IgG anti-double-stranded DNA (dsDNA) antibodies was significantly lower in p40-lpr(cg). Anti-dsDNA antibodies decreased in Th1-dependent IgG2a but increased in the Th2-dependent IgG1 subclass significantly in p40-lpr(cg). Proteinuria, glomerulonephritis, and survival were only marginally ameliorated in p40-lpr(cg). The results suggest that excess p40 production in vivo may suppress Th1 responses in autoantibody and IFN-gamma production but lead to minimal improvement of clinical manifestations of autoimmune disease in this mouse model.

Molecular mechanisms of the decision between life and death: regulation of apoptosis by apoptosis signal-regulating kinase 1.

Coordination and balance between cell survival and apoptosis is crucial for normal development and homeostasis of multicellular organisms. Defects in control of this balance may contribute to a variety of diseases including cancer, autoimmune and neurodegenerative conditions. Although a large number of pro- and anti-apoptotic factors acting for or against the final death event have been and are being discovered at an extraordinary pace with the recent progress in this area, the molecular mechanisms determining whether a cell lives or dies are not fully understood. Phosphorylation and dephosphorylation of intracellular effector molecules are the most common and important regulatory mechanisms in signal transduction and control a variety of cellular events from cell growth to apoptosis. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein (MAP) kinase kinase kinase family, which activates both the SEK1-JNK and MKK3/6-p38 MAP kinase pathways and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. This review provides recent findings on the molecular mechanisms which determine cell fate such as survival, proliferation, differentiation or apoptosis, with special focus on the regulatory mechanisms of ASK1-mediated apoptosis.

Metallothionein expression and apoptosis in pregnancy-dependent and -independent mouse mammary tumors.

BACKGROUND: A metal binding protein, metallothionein (MT), may be involved in the regulation of carcinogenesis and apoptosis in addition to various physiological processes. MATERIALS AND METHODS: MT and sex hormone receptors (estrogen and progesterone receptors, ER and PR) expressions, and apoptosis were investigated immunohistochemically in pregnancy-dependent (PDMT) and -independent mammary tumors (PIMT) in GR/A mice in order to evaluate the possible role of MT in the genesis and regression of tumors. RESULTS: In PDMT and PIMT, MT was localized in the nucleus and/or cytoplasm of tumor cells. In PDMT, MT expression and apoptotic figures were increased during the regression period after parturition. MT and ER expressions in PIMT were approximately the same as those in the growing PDMT, while PR expression was lower in PIMT than in the PDMT. A significant correlation was observed between MT expression and apoptosis in PDMT, but not in PIMT. CONCLUSION: The present study suggests that MT involvement in the PDMT regression is associated with apoptosis.

Characterization of mouse mammary tumour virus-induced migration of lymphoid cells into lymph nodes.

We previously found that Mtv-2+ lymph nodes (LN) implanted into Mtv-2- mice underwent marked hyperplasia owing to the influx of lymphocytes. LN grafts infected with exogenous mouse mammary tumour viruses (MMTV), MMTV(FM) transmitted by FM mice and MMTV-2 produced by Mtv-2, also swelled in MMTV-free recipients. Mtv-3 and Mtv-7 also displayed this capability. Mtv-2-induced LN hyperplasia was earlier in onset and greater in extent when major histocompatibility complex (MHC) class II I-E was expressed than unexpressed. Mtv-3-induced LN hyperplasia was suppressed completely by Mtv-3 from a different mouse strain and partially by Mtv-6 slightly different from Mtv-3 in superantigen (SAg) Vbeta specificity. LN hyperplasia occurred bidirectionally in LN transplantation between mice carrying Mtv-2 and Mtv-3, which are different SAg Vbeta specificity. LN hyperplasia induced by MMTV-2 carrying SAg responsive to Vbeta14 alone and MMTV(FM) carrying SAg responsive to Vbeta14 and Vbeta8.2 was completely but partially suppressed by MMTV(FM) and MMTV-2, respectively. CD4+ T cells were essential for MMTV-induced LN hyperplasia. LN in situ also underwent significant hyperplasia when infected with MMTV. Thus, MMTV SAg may entice circulating lymphocytes into lymphoid organs and contribute to more efficient dissemination MMTV in vivo. Secondary lymphoid tissue chemokine (SLC) may not be directly involved in this event.

Antitumor effects of a new lipophilic platinum compound, trans-bis(n-valerato)(1R,2R-cyclohexanediamine)(oxalato)platinum(IV), in mice.

A new lipophilic platinum (Pt) compound, trans-bis(n-valerato)(1R,2R-cyclohexanediamine)(oxalato)Pt(lV) (C5-OHP), was compared with cisplatin (CDDP) by intraperitoneal injection for antitumor activities against 3 different mouse models, L1210 leukemia, LMFS sarcoma with high metastatic potential and spontaneous mammary tumor. C5-OHP cured both CDDP-sensitive and -resistant L1210 leukemia frequently. CDDP did not cure the leukemia, although it prolonged the survival of diseased mice significantly. C5-OHP cured early and advanced LMFS sarcomas more frequently than CDDP. Cured mice had acquired antitumor immunity, indicating the pivotal role of the immune system in induction of the cure of tumors. It is likely tllat C5-OHP can eradicate tumor cells more effectively than CDDP by virtue of its weaker suppressive effects on the immune system. C5-OHP but not CDDP could cure mammary tumors. C5-OHP manifested a curative effect against LMFS tumors by oral route. These results indicate that C5-OHP is a promising anticancer agent worthy of clinical trial.

ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis.

Apoptosis signal-regulating kinase (ASK) 1 is activated in response to various cytotoxic stresses including TNF, Fas and reactive oxygen species (ROS) such as H(2)O(2), and activates c-Jun NH(2)-terminal kinase (JNK) and p38. However, the roles of JNK and p38 signaling pathways during apoptosis have been controversial. Here we show that by deleting ASK1 in mice, TNF- and H(2)O(2)-induced sustained activations of JNK and p38 are lost in ASK1(-/-) embryonic fibroblasts, and that ASK1(-/-) cells are resistant to TNF- and H(2)O(2)-induced apoptosis. TNF- but not Fas-induced apoptosis requires ROS-dependent activation of ASK1-JNK/p38 pathways. Thus, ASK1 is selectively required for TNF- and oxidative stress-induced sustained activations of JNK/p38 and apoptosis.

Fas/Apo-1 (CD95)-mediated apoptosis of neutrophils with Fas ligand (CD95L)-expressing tumors is crucial for induction of inflammation by neutrophilic polymorphonuclear leukocytes associated with antitumor immunity.

To address how FasL-expressing tumors induce neutrophil emigration and abrogate tumorigenicity, we investigated the behavior of FasLcDNA-transfected hepatoma MH134 (G2) cells injected into wild-type (+) mice, lpr(cg)/lpr(cg) (lpr(cg)) mice with death domain (DD)-mutated Fas, and gld/gld lpr/lpr (gld/lpr) mice with defects in FasL/Fas. G2 cells were eradicated after extensive infiltration of neutrophils around them in + mice but formed tumors without such infiltration in lpr(cg) and gld/lpr mice. Abundant cell debris suggestive of apoptosis of infiltrating neutrophils was found among G2 tumor cells in + mice but a few neutrophils infiltrating among G2 cells were intact in lpr(cg) and gld/lpr mice. Collectively, these results indicate the crucial role of Fas DD in Fas-mediated apoptosis of neutrophils and suggest that apoptosis of neutrophils with FasL-expressing tumors may trigger the extensive infiltration of neutrophils, resulting in violent inflammation and ultimately in the eradication of tumor cells.

Alleviation of renal disease and lymphadenopathy in MRL-Fasp(lrcg)/Fas(lprcg) (MR-lpr(cg)) mice neonatally infected with mouse mammary tumor virus encoding superantigen strongly reactive with TCR Vbeta8.2 element.

Mouse mammary tumor virus transmitted by FM mice (FM-MMTV) encodes a superantigen (SAg) characterized by strong reactivity with TCR Vbeta8.2 element and broad spectrum of Vbeta reactivity. To investigate what effects the expression in vivo of FM-MMTV SAg exhibits on the course of the disease in a lupus-prone model, MRL/MpJ-Fas(lprcg)/Fas(lprcg) (MRL-lpr9cg) mice, neonatally FM-MMTV-infected MRL-lprcg(MMTV) and uninfected MRL-lpr(cg) mice were compared for various disease parameters. In MRL-lprcg(MMTV), survival was significantly prolonged, glomerulonephritis, proteinuria, and lymphadenopathy were clearly ameliorated, and the production of serum immunoglobulin G (IgG), complement-activating IgG2a, and cryogenic IgG3 autoantibodies, which are thought to be pathogenic to kidneys, and circulating immune complexes (IC), and glomerular IC deposition were significantly suppressed. FM-MMTV infection deleted Vbeta8.2+ cells by about 90% and Vbeta14+ cells less efficiently in all of the CD4+, CD8+, and B220+ CD4- CD8- or double-negative (DN) T-cell populations, and Vbeta8.1+ cells in the CD4+ population but not in the others. Similar deletion profiles of CD8+ and DN T cells support that DN T cells are derived from the CD8 lineage. The results imply that the specific regulation of the immune system with viral SAg has a potential for development of an attractive immunomodulatory therapy of autoimmune diseases.

A pivotal role of cell-bound but not soluble CD4 molecules in full development of lupus-like manifestations in MRL-Fas(lprcg)/Fas(lprcg) mice.

The role of CD4 molecules in the autoimmune and lymphoproliferative syndrome caused by murine Fas mutations was studied using the novel systemic lupus erythematosus (SLE) model, MRL-Fas(lpr(cg))/Fas(lprcg) (MRL-lpr(cg)) mice, in combination with the novel mutant CD4 gene producing soluble CD4 (sCD4) instead of membrane-bound CD4 (mCD4). For this purpose, various autoimmune manifestations were compared among MRL-lpr(cg) mice homozygous (CD4slprcg), heterozygous (CD4s/mlpr(cg)), and wild-type (CD4mlpr(cg)) for the CD4 mutation. The mortality, glomerulonephritis, proteinuria, and lymphadenopathy were significantly ameliorated in CD4slprcg compared with CD4mlpr(cg) and CD4s/mlpr(cg) mice, both being comparable in these clinical characteristics. In parallel with the clinical improvement, the serum levels of immunoglobulin, anti-DNA antibodies, anti-nuclear antibodies and immune complexes, and the extent of glomerular immune deposition, were significantly lower in the former. The results indicate that mCD4 is important and can not be replaced by sCD4 in full development of SLE-like manifestations, and suggest that CD4+ T cells may aggravate the autoimmune disease by stimulating autoreactive B cells to produce autoantibodies through their helper activity in Fas mutant models. The sCD4 levels in the serum and spleen elevated with the increased accumulation of B220+CD4-CD8- (double-negative (DN)) T cells in CD4slpr(cg) mice. This, together with the significantly milder lymphadenopathy associated with lower DN T cell contents in CD4slpr(cg) than CD4mlpr(cg) mice, implies that some of abnormal DN T cells may be derived from cells of the CD4 lineage.

Impaired delayed-type hypersensitivity response in mutant mice secreting soluble CD4 without expression of membrane-bound CD4.

Delayed-type hypersensitivity (DTH) is an important in vivo manifestation of cell-mediated immunity. We examined the DTH response to methylated bovine serum albumin of a novel mutant strain of mice that have soluble CD4 (sCD4) in their circulation without expression of CD4 on the cell surface. The DTH response of the mutant mice was severely impaired, although the response of CD4 knockout (KO) mice, generated by homologous recombination, was comparable to that of wild-type mice. The response of the mutant mice was restored by the neutralization of sCD4 with anti-CD4, and that of CD4KO mice was markedly reduced by the implantation of a diffusion chamber containing sCD4 cDNA transfectant cells. The restored DTH response of the mutant mice treated with anti-CD4 was abolished by treatment with anti-interferon-gamma (IFN-gamma). IFN-gamma production by CD4 mutant and CD4KO mice was consistent with their DTH response and inversely related to the presence of sCD4 in their circulation, indicating that sCD4 impairs the DTH response by blocking the production of IFN-gamma in our mutant mice. These results raise the possibility that sCD4 could impair cell-mediated immunity. Our mutant mice would provide a useful tool with which to analyse the mechanisms of the DTH reaction.

Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation.

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.

Metallothionein expression in transplantable mouse mammary tumors.

BACKGROUND: The metal binding protein, metallothionein (MT) is involved in various physiological processes. In various human tumors, moreover, MT is reported to play an important role in carcinogenesis. MATERIALS AND METHODS: MT expression was investigated immunohistochemically and chromatographically in a transplantable pregnancy-dependent mouse mammary tumor (TPDMT-4) and related autonomous tumor sublines (T4-OI320 and -OI320CY). RESULTS: All tumor lines showed MT expression in the nucleus and/or cytoplasm of tumor cells. However, adenoacanthomatous T4-OI320 whose structure was different histopathologically from the remaining adenocarcinomas showed over-expression of MT in the basal layer of squamous metaplastic nodules. Cell proliferation activity estimated by the BrdU labeling method (BrdU index) was highest in the squamous nodules and correlated with the degree of MT expression (r = 0.96). By contrast, the BrdU index in T4-OI320CY was inversely correlated with the MT index (r = -0.91). CONCLUSIONS: The present investigation suggests expression of isotype MT in the transplantable mouse mammary tumors which may be involved in the carcinogenesis.

Immunological characterization of C3H mice congenic for Fas(lprcg), C3h/HeJ-Fas(lprcg)/Fas(lprcg).

Fas(lpr) (lpr) and Fas(lprcg) (lpr(cg)) are allelic mutations of the Fas gene that is involved in apoptosis or programmed cell death. Lpr greatly reduces the expression of functional Fas and lpr(cg) expresses the death domain-disabled, non-functional Fas on the cell surface. C3H/HeJ mice congenic for lpr(cg) (C3H-lpr(cg)) were established and compared with C3H/HeJ-lpr/lpr (C3H-lpr) mice for their immunological and pathological features. Lymphadenopathy, splenomegaly, development of CD4- CD8- B220+ or double-negative (DN) T cells, renal pathology, and lymphoid cell infiltration in the lung and liver were not significantly different between C3H-lpr(cg) and C3H-lpr mice. Noticeably, however, the production of serum immunoglobulin, autoantibodies against double-strand DNA and serum immune complexes were significantly lower in C3H-lpr(cg) than in C3H-lpr mice. The results indicate that the death signal through the death domain of Fas is responsible for lymphoproliferation due to the accumulation of DN T cells and suggest that the region of Fas outside the death domain may be involved in autoantibody production. The newly-developed congenic C3H-lpr(cg) mice will provide a powerful tool for research into the function of Fas apart from apoptosis.

Role of IL-16 in delayed-type hypersensitivity reaction.

Interleukin (IL)-16 is a chemoattractant cytokine for CD4(+) leukocytes. Because delayed-type hypersensitivity (DTH) reaction is mediated by T helper 1 (Th1) cells and CD4(+) T cells can be chemoattracted by IL-16, we have investigated the involvement of IL-16 in the DTH reaction. Immunohistochemical analysis revealed the IL-16 expression in infiltrating cells and epithelial cells in the DTH footpads. The IL-16 expression was also detected intracellularly in the infiltrating cells. In addition, markedly increased production of IL-16 was detected in the DTH footpad extracts, but not in the control footpad extracts, by an enzyme-linked immunosorbent assay and also by Western blot analysis. The DTH footpad extracts exhibited a strong chemoattractant activity toward splenic T cells, which was significantly inhibited by the inclusion of neutralizing monoclonal antibody (mAb) against IL-16 in the migration assay. Furthermore, treatment of sensitized mice in vivo with the anti-IL-16 neutralizing mAb significantly suppressed the footpad swelling induced by an antigen challenge, together with decreased infiltration of leukocytes including not only CD4(+) T cells but also CD8(+) T cells and macrophages into the DTH footpads. Decreased production of macrophage inflammatory protein 1alpha was also observed in the DTH footpad extracts by the mAb treatment. These results suggest that IL-16 plays an important role in the recruitment of leukocytes-presumably including antigen-specific Th1 cells, which secrete cytokines and chemokines mediating the following hypersensitivity reaction after activation by the interaction with Langerhans cells carrying the antigen-for the elicitation of DTH response. (Blood. 2000;95:2869-2874)

The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules.

T cell homing to peripheral lymph nodes (PLNs) is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial tethering and rolling via L-selectin, firm adhesion of T cells requires rapid upregulation of lymphocyte function-associated antigen 1 (LFA-1) adhesiveness by a previously unknown pathway that activates a Galpha(i)-linked receptor. Here, we used intravital microscopy of murine PLNs to study the role of thymus-derived chemotactic agent (TCA)-4 (secondary lymphoid tissue chemokine, 6Ckine, Exodus-2) in homing of adoptively transferred T cells from T-GFP mice, a transgenic strain that expresses green fluorescent protein (GFP) selectively in naive T lymphocytes (T(GFP) cells). TCA-4 was constitutively presented on the luminal surface of HEVs, where it was required for LFA-1 activation on rolling T(GFP) cells. Desensitization of the TCA-4 receptor, CC chemokine receptor 7 (CCR7), blocked T(GFP) cell adherence in wild-type HEVs, whereas desensitization to stromal cell-derived factor (SDF)-1alpha (the ligand for CXC chemokine receptor 4 [CXCR4]) did not affect T(GFP) cell behavior. TCA-4 protein was not detected on the luminal surface of PLN HEVs in plt/plt mice, which have a congenital defect in T cell homing to PLNs. Accordingly, T(GFP) cells rolled but did not arrest in plt/plt HEVs. When TCA-4 was injected intracutaneously into plt/plt mice, the chemokine entered afferent lymph vessels and accumulated in draining PLNs. 2 h after intracutaneous injection, luminal presentation of TCA-4 was detectable in a subset of HEVs, and LFA-1-mediated T(GFP) cell adhesion was restored in these vessels. We conclude that TCA-4 is both required and sufficient for LFA-1 activation on rolling T cells in PLN HEVs. This study also highlights a hitherto undocumented role for chemokines contained in afferent lymph, which may modulate leukocyte recruitment in draining PLNs.

The role of chemokines in the microenvironmental control of T versus B cell arrest in Peyer's patch high endothelial venules.

Chemokines have been hypothesized to contribute to the selectivity of lymphocyte trafficking not only as chemoattractants, but also by triggering integrin-dependent sticking (arrest) of circulating lymphocytes at venular sites of extravasation. We show that T cells roll on most Peyer's patch high endothelial venules (PP-HEVs), but preferentially arrest in segments displaying high levels of luminal secondary lymphoid tissue chemokine (SLC) (6Ckine, Exodus-2, thymus-derived chemotactic agent 4 [TCA-4]). This arrest is selectively inhibited by functional deletion (desensitization) of CC chemokine receptor 7 (CCR7), the receptor for SLC and for macrophage inflammatory protein (MIP)-3beta (EBV-induced molecule 1 ligand chemokine [ELC]), and does not occur in mutant DDD/1 mice that are deficient in these CCR7 ligands. In contrast, pertussis toxin-sensitive B cell sticking does not require SLC or MIP-3beta signaling, and occurs efficiently in SLC(low/-) HEV segments in wild-type mice, and in the SLC-negative HEVs of DDD/1 mice. Remarkably, sites of T and B cell firm adhesion are segregated in PPs, with HEVs supporting B cell accumulation concentrated in or near follicles, the target domain of most B cells entering PPs, whereas T cells preferentially accumulate in interfollicular HEVs. Our findings reveal a fundamental difference in signaling requirements for PP-HEV recognition by T and B cells, and describe an unexpected level of specialization of HEVs that may allow differential, segmental control of lymphocyte subset recruitment into functionally distinct lymphoid microenvironments in vivo.