[show abstract][hide abstract] ABSTRACT: Monocytes recruited from the blood are key contributors to the nature of an immune response. While monocyte recruitment in a subset of immunopathologies has been well studied and largely attributed to the chemokine monocyte chemoattractant protein (MCP)-1, mechanisms mediating such recruitment to other sites of inflammation remain elusive. Here, we showed that localized inflammation resulted in an increased binding of monocytes to perifollicular high endothelial venules (HEVs) of lymph nodes draining a local inflammatory site. Quantitative PCR analyses revealed the upregulation of many chemokines in the inflamed lymph node, including MCP-1 and MIG. HEVs did not express detectable levels of MCP-1; however, a subset of HEVs in inflamed lymph nodes in wild-type (but not tumor necrosis factor [TNF] null mice) expressed MIG and this subset of HEVs preferentially supported monocyte binding. Expression of CXCR3, the receptor for MIG, was detected on a small subset of peripheral blood monocytes and on a significant percentage of recruited monocytes. Most importantly, in both ex vivo and in vivo assays, neutralizing anti-MIG antibodies blocked monocyte binding to inflamed lymph node HEVs. Together, these results suggest that the lymph node microenvironment can dictate the nature of molecules expressed on HEV subsets in a TNF-dependent fashion and that inflammation-induced MIG expression by HEVs can mediate monocyte recruitment.
Journal of Experimental Medicine 12/2001; 194(9):1375-84. · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mice without secreted TNF but with functional, normally regulated and expressed membrane-bound TNF (memTNF(Delta/Delta) mice) were created by knocking-in the uncleavable Delta 1-9,K11E TNF allele. In contrast to TNF-deficient mice (TNF(-/-)), memTNF supported many features of lymphoid organ structure, except generation of primary B cell follicles. Splenic chemokine expression was near normal. MemTNF-induced apoptosis was mediated through both TNF-R1 and TNF-R2. That memTNF is suboptimal for development of inflammation was revealed in experimental autoimmune encephalomyelitis. Disease severity was reduced in memTNF(Delta/Delta) mice relative to wild-type mice, and the nature of spinal cord infiltrates resembled that in TNF(-/-) mice. We conclude that memTNF supports many processes underlying lymphoid tissue structure, but secreted TNF is needed for optimal inflammatory lesion development.
[show abstract][hide abstract] ABSTRACT: Lymphotoxin (LT)-alpha, a member of the TNF family, is recognized as an important mediator in different aspects of lymphoid organ development. Targeted disruption of this molecule resulted in a substantial reduction in the proportion of alphaEbeta7-integrin(high) CD8+ T cells detectable in peripheral lymphoid organs. This defect, however, was not observed on mature CD4-CD8+ thymocytes. To determine whether this was due to downregulation of beta7-integrin expression by peripheral CD8+ T cells or a failure of thymic emigration of CD8+ beta7-integrin(high) T cells, beta7-integrin was examined on recent thymic emigrants (RTE). When analysed within 16 h after leaving the thymus CD4-CD8+ RTE in both LT-alpha-/- and wild type (wt) mice remained beta7-integrin(high) and were indistinguishable. However, within 3-5 days, emigration loss of beta7-integrin became evident in LT-alpha-/- mice. Despite this loss, the proportion of thymically derived alphabetaTCR+ T-cell populations in the intestinal epithelium, an important target tissue of CD8+ alphaEbeta7-integrin(high) T cells, was increased in the absence of LT-alpha. In contrast, B cells were detectable only rarely in the intestinal tissue of LT-alpha-/- mice. The expression of E-Cadherin remained unchanged. These results indicate that a LT-alpha-dependent process maintains a high level of alphaEbeta7-integrin expression by peripheral CD8+ T cells, and with this control mechanism LT-alpha may help to regulate CD8+ T-cell numbers in the tissues.
Immunology and Cell Biology 09/2001; 79(4):323-31. · 3.93 Impact Factor
[show abstract][hide abstract] ABSTRACT: In normal spleen, most recirculating naïve IgM+IgDhi B cells are located within primary follicles and mantle zones of secondary follicles. By contrast, the marginal zone contains a heterogeneous population of IgMhiIgDlo/- B cells that are mostly non-recirculating. Although these are dynamic populations they are maintained at a constant size, the fundamental homeostatic mechanisms remain uncertain. One possibility is that the presence and turnover of each of the B cell populations is dependent on their location within discrete splenic compartments. To investigate this, we have characterized immature, non-recirculating, mature recirculating, marginal zone and B-1 cell populations in TNF-/- and TNF/lymphotoxin(LT)-alpha-/- mice that have disorganized splenic architecture. Labelling with 5-bromo-2'-deoxyuridine revealed that turnover of B cells in TNF-/- mice is normal, but is diminished in TNF/LT-alpha-/- mice. The recirculating B cell populations in both mutant strains are normal in proportion and phenotype. Marginal zone B cells are not seen in TNF/LT-alpha-/- mice, but this population appears normal in TNF-/- mice, even though they lack germinal centres. These findings indicate that peripheral B cell subsets can be established and maintained independently of normal follicular architecture.
Immunology and Cell Biology 03/2001; 79(1):54-61. · 3.93 Impact Factor
[show abstract][hide abstract] ABSTRACT: OX2 (CD200) is a broadly expressed membrane glycoprotein, shown here to be important for regulation of the macrophage lineage. In mice lacking CD200, macrophage lineage cells, including brain microglia, exhibited an activated phenotype and were more numerous. Upon facial nerve transection, damaged CD200-deficient neurons elicited an accelerated microglial response. Lack of CD200 resulted in a more rapid onset of experimental autoimmune encephalomyelitis (EAE). Outside the brain, disruption of CD200-CD200 receptor interaction precipitated susceptibility to collagen-induced arthritis (CIA) in mice normally resistant to this disease. Thus, in diverse tissues OX2 delivers an inhibitory signal for the macrophage lineage.
[show abstract][hide abstract] ABSTRACT: DAP12 is an ITAM-bearing membrane adaptor molecule implicated in the activation of NK and myeloid cells. In mice rendered DAP12 deficient by targeted gene disruption, lymphoid and myeloid development was apparently normal, although the activating Ly49 receptors on NK cells were downregulated and nonfunctional. To analyze the consequences of DAP12 deficiency in vivo, we examined the susceptibility of DAP12-/- mice to experimental autoimmune encephalomyelitis (EAE). DAP12-/- mice were resistant to EAE induced by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide. Resistance was associated with a strongly diminished production of IFNgamma by myelin-reactive CD4+ T cells due to inadequate T cell priming in vivo. These data suggest that DAP12 signaling may be required for optimal antigen-presenting cell (APC) function or inflammation.
[show abstract][hide abstract] ABSTRACT: The OX2 membrane glycoprotein (CD200) is expressed on a broad range of tissues including lymphoid cells, neurons, and endothelium. We report the characterization of an OX2 receptor (OX2R) that is a novel protein restricted to cells of the myeloid lineage. OX2 and its receptor are both cell surface glycoproteins containing two immunoglobulin-like domains and interact with a dissociation constant of 2.5 microM and koff 0.8 s(-1), typical of many leukocyte protein membrane interactions. Pervanandate treatment of macrophages showed that OX2R could be phosphorylated on tyrosine residues. Blockade of the OX2-OX2R interaction with an OX2R mAb exacerbated the disease model experimental allergic encephalomyelitis. These data, together with data from an OX2-deficient mouse (R. M. Hoek et al., submitted), suggest that myeloid function can be controlled in a tissue-specific manner by the OX2-OX2R interaction.
[show abstract][hide abstract] ABSTRACT: Lymphoid follicles are B-cell-rich compartments of lymphoid organs that function as sites of B-cell antigen encounter and differentiation. CXC chemokine receptor-5 (CXCR5) is required for B-cell migration to splenic follicles, but the requirements for homing to B-cell areas in lymph nodes remain to be defined. Here we show that lymph nodes contain two types of B-cell-rich compartment: follicles containing follicular dendritic cells, and areas lacking such cells. Using gene-targeted mice, we establish that B-lymphocyte chemoattractant (BLC/BCA1) and its receptor, CXCR5, are needed for B-cell homing to follicles in lymph nodes as well as in spleen. We also find that BLC is required for the development of most lymph nodes and Peyer's patches. In addition to mediating chemoattraction, BLC induces B cells to up-regulate membrane lymphotoxin alpha1beta2, a cytokine that promotes follicular dendritic cell development and BLC expression, establishing a positive feedback loop that is likely to be important in follicle development and homeostasis. In germinal centres the feedback loop is overridden, with B-cell lymphotoxin alpha1beta2 expression being induced by a mechanism independent of BLC.
[show abstract][hide abstract] ABSTRACT: TNF receptor-ligand interactions and CD95 (Fas / APO-1) have been demonstrated to be involved in activation-induced death of mature T cells. Here, we examined the role of these molecules in the murine model of lymphocytic choriomeningitis virus (LCMV) infection using LCMV TCR transgenic (tg) mice lacking TNF, TNF receptor I (TNFR1), CD95 or both TNFR1 and CD95. This report demonstrates that neither TNF receptor-ligand interactions nor CD95 was required for down-regulation of LCMV-specific CD8 T cells following acute LCMV infection in vivo. Even LCMV-specific CD8 T cells lacking both TNFR1 and CD95 molecules declined after the acute phase of the infection with normal kinetics. Furthermore, peripheral deletion of LCMV-specific CD8 T cells induced by LCMV peptide injection or by adoptive transfer of tg spleen cells expressing the corresponding LCMV epitope was not impaired in mice lacking TNF, TNFR1 and / or CD95. Our data speak against an indispensable role of these molecules in antigen-induced apoptosis of CD8 T cells in vivo and suggest that T cell homeostasis after antigen challenge is controlled by additional mechanisms.
European Journal of Immunology 03/2000; 30(2):678-82. · 4.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: TNF, lymphotoxin (LT) and their receptors are expressed constitutively in the thymus. It remains unclear whether these cytokines play a role in normal thymic structure or function. We have investigated thymocyte differentiation, selection and thymic organogenesis in gene targeted mice lacking LTalpha, TNF, or both (TNF/LTalpha-/-). The thymus was normal in TNF/LTalpha-/- mice with regard to cell yields and stromal architecture. Detailed analysis of alphabeta and gammadelta T cell-lineage thymocyte subsets revealed no abnormalities, implying that neither TNF nor LT play an essential role in T cell differentiation or positive selection. The number and distribution of thymic CD11c+ dendritic cells was also normal in the absence of both TNF and LTalpha. A three-fold increase in B cell numbers was observed consistently in the TNF/LTalpha-/- thymus. This phenotype was due entirely to the LTalpha deficiency and associated with changes in the hemopoietic compartment, rather than the thymic stromal compartment of LTalpha-/- mice. Finally, specific Vbeta8+ T cell deletion within the thymus following intrathymic injection of staphylococcal enterotoxin B (SEB) was TNF/LT independent. Thus, despite the presence of these cytokines and their receptors in the normal thymus, there appears no essential role for either TNF or LT in development of organ structure or for those processes associated with T cell repertoire selection.
[show abstract][hide abstract] ABSTRACT: Tumor necrosis factor (TNF) and Fas ligand (FasL) play major roles in the homeostasis of the peripheral immune system. This becomes dramatically obvious in the absence of a functional FasL. Mice with such a deficiency develop a profound lymphadenopathy, splenomegaly, hypergammaglobulinemia, and strain-dependent systemic autoimmune disease, and succumb to premature death. It is consequently termed generalized lymphoproliferative disorder (gld). By contrast, TNF deficiency alone does not result in a striking phenotype. Thus, we sought to determine what role TNF might play in contributing to the gld phenotype by creating C57BL/6.gld.TNF(-/-) mice. Contrary to the expected outcome, mice deficient for both FasL and TNF had a substantially milder gld phenotype with regard to mortality, lymphoaccumulation, germinal center formation, and hypergammaglobulinemia. To confirm these data in a strain highly permissive for the phenotype, C3H/HeJ.gld and C3H.HeJ.lpr mice were treated with a TNF-specific monoclonal antibody. This transient neutralization of TNF also resulted in a significantly attenuated lymphoproliferative phenotype. We conclude that TNF is necessary for the full manifestation of the lymphoproliferative disorder, in particular playing a critical role in lymphoaccumulation. Most importantly, absence of TNF protects gld mice against premature death.
Journal of Experimental Medicine 02/2000; 191(1):89-96. · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have evaluated the NK cell antitumor activity in lymphotoxin (LT)-deficient mice. Both NK cell-mediated tumor rejection and protection from experimental metastases were significantly compromised in LT-alpha-deficient mice. Analysis of LT-alpha-deficient mice revealed that the absolute number of alphabetaTCR- NK1.1+ NK cells was reduced in bone marrow and thymus, but with overall proportional decreases in other hemopoietic organs. In addition, the antitumor potential of alphabetaTCR- NK1.1+ cells, as determined by their lytic capacity and perforin expression, was reduced 1.5- to 3-fold in LT-alpha-deficient mice, as compared with wild-type mice. Combined defects in NK cell development and effector function contribute to compromised NK cell antitumor function in LT-alpha-deficient mice.
The Journal of Immunology 09/1999; 163(3):1350-3. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mice infected with an adenovirus mutant in which the E3 region is deleted, including TNF-resistance genes, develop fatal liver pathology within 3-4 days after infection. At least 10-fold more wild-type virus was needed to cause comparable pathology. These results indicate that the E3 region is critically involved in modulating the pathogenesis of adenovirus infection and that TNF may play a role in liver damage. To explore the latter possibility, the course of disease was examined in infected mice lacking TNFR-I and/or TNFRII, TNF only, or both TNF and lymphotoxin-alpha. Only mice lacking both TNFRI and TNFRII were protected from the lethal affects of the mutant adenovirus. Mice deficient in TNF or TNF and lymphotoxin-alpha displayed the fatal pathology. This outcome is consistent with the existence of another related ligand that binds TNFRI/II to mediate liver damage during infection with this mutant.
The Journal of Immunology 09/1999; 163(3):1516-20. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: 5,6-Dimethylxanthenon-4-acetic acid (DMXAA) is a new antitumor drug currently undergoing clinical trial. Administration of DMXAA to mice with tumors leads to cessation of tumor blood flow and the onset of tumor hemorrhagic necrosis, accompanied by the production of the cytokine tumor necrosis factor (TNF). Previous studies have shown that DMXAA induces both tumor and host cells to synthesize TNF and that induced intratumoral TNF production correlates with the antitumor activity of DMXAA. To explore the hypothesis that TNF production by tumor cells contributed to the induction of hemorrhagic necrosis by DMXAA, TNF-/- (C57Bl/6 background) mice were used as recipients for the s.c. implantation of (TNF positive) colon 38 adenocarcinoma. Tumors removed 24 h after treatment with DMXAA (66 or 100 micromol/kg) were found to be hemorrhagic and necrotic. Cells expressing TNF mRNA in tumors removed 2 h after treatment with DMXAA (160 micromol/kg) were found by in situ hybridization to be comparable in frequency and distribution with those in tumors from C57Bl/6 TNF-positive mice. However, the amount of TNF protein extracted from tumors from TNF knockout mice was lower than that from TNF-positive mice. Spleen and liver tissue from TNF knockout mice, in contrast to that from TNF-positive mice, produced no TNF mRNA. TNF protein was undetectable in liver and spleen tissue from TNF knockout mice, but was evident in tissue from TNF-positive mice. These results confirm that DMXAA has the novel ability of inducing tumors to synthesize TNF in situ.
Cancer Research 08/1999; 59(14):3304-7. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: TNF and lymphotoxin-alpha (LT alpha) may act at various stages of the host response to Mycobacterium tuberculosis. To dissect the effects of TNF independent of LT alpha, we have used C57BL/6 mice with a disruption of the TNF gene alone (TNF-/-). Twenty-one days following aerosol M. tuberculosis infection there was a marked increase in the number of organisms in the lungs of TNF-/- mice, and by 28-35 days all animals had succumbed, with widespread dissemination of M. tuberculosis. In comparison with the localized granulomas containing activated macrophages and T cells in lungs and livers of C57BL/6 wild-type (wt) mice, cellular infiltrates in TNF-/- mice were poorly formed, with extensive regions of necrosis and neutrophilic infiltration of the alveoli. Phenotypic analysis of lung homogenates demonstrated similar numbers of CD4+ and CD8+ T cells in TNF-/- and wt mice, but in TNF-deficient mice the lymphocytes were restricted to perivascular and peribronchial areas rather than colocated with macrophages in granulomas. T cells from TNF-/- mice retained proliferative and cytokine responses to purified protein derivative, and delayed-type hypersensitivity to purified protein derivative was demonstrable. Macrophages within the lungs of TNF-/- and wt mice showed similar levels of MHC class II and inducible nitric oxide synthase expression, and levels of serum nitrite were comparable. Thus, the enhanced susceptibility of TNF-/- is not compensated for by the presence of LT alpha, and the critical role of TNF is not in the activation of T cells and macrophages but in the local organization of granulomas.
The Journal of Immunology 04/1999; 162(6):3504-11. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: To evaluate the importance of the proinflammatory cytokines TNF and lymphotoxin-alpha (LT alpha) in an experimental model of Staphylococcus aureus sepsis and arthritis, we used TNF/LT alpha-double-deficient mice raised on the C57BL/6 background. Mice were i.v. inoculated with a toxic shock syndrome toxin-1 (TSST-1)-producing S. aureus strain, LS-1. Intravenous inoculation of a high dose of bacteria (1 x 10(7)/mouse) resulted in 67% mortality in TNF/LT alpha-deficient mice, whereas none of the controls died (p = 0.009). Those results correlated to a significantly decreased phagocytosis in vitro and inefficient bacterial clearance in vivo in mice lacking capacity to produce TNF/LT alpha. Thus, at day 6 after inoculation, S. aureus could not be found in the bloodstream of controls, but bacteremia developed in all TNF/LT alpha-deficient mice examined (p = 0.02). Interestingly, upon infection with a lower dose of staphylococci (3 x 10(6)/mouse) the mortality was overall low, but the frequency of arthritis was clearly higher in the wild-type group as compared with the TNF/LT alpha-deficient mice (40% vs 13%). Histopathologic examination revealed a lower frequency of synovitis (38% vs 90%, p < 0.05) and erosivity (25% vs 60%, NS) in TNF/LT alpha-deficient mice as compared with wild-type counterparts. Our results show the importance of TNF/LT alpha in defense against systemic S. aureus infections and point out the detrimental role of these cytokines as mediators of inflammatory response in S. aureus arthritis.
The Journal of Immunology 01/1999; 161(11):5937-42. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mouse CD8+ CTL reactive with an H-2Db presented 9-mer peptide of the human papilloma virus 16 (HPV-16) protein E749-57 (RAHYNIVTF) were generated from the splenocytes of wild-type C57BL/6 (B6), B6.perforin-deficient, B6.gld or B6.TNF-deficient mice. In short-term (4 h) assays, CTL from B6, B6.TNF-deficient and B6.gld mice displayed peptide-specific perforin- and/or Fas ligand (FasL)-mediated lysis of E7-transfected mouse RMA lymphoma cells (RMA-E7) or E749-57 peptide-pulsed RMA-S cells, while CD8+ CTL from B6.perforin-deficient mice lysed via FasL exclusively. Rapid and efficient lysis of syngeneic bystander B6 spleen T cell blasts by B6, B6.TNF-deficient or B6.perforin-deficient antigen-activated CTL was mediated apparently exclusively by a FasL/Fas mechanism. By contrast CTL from B6.gld mice did not mediate rapid bystander lysis of B6 blasts. Rather B6.gld CTL delivered delayed bystander lysis after 36-48 h that was mediated by TNF. TNF-mediated bystander lysis of syngeneic blasts appeared to be independent of class I molecules and was mediated at least in part by soluble TNF. By contrast, there was no evidence that soluble FasL-mediated bystander lysis. For the first time, these data indicate that CD8+ CTL may use FasL or TNF in a kinetically and physically distinct fashion to mediate bystander killing.
European Journal of Immunology 01/1999; 28(12):4162-9. · 4.97 Impact Factor