WT Bone Marrow Research Articles

Requirement of IL-17 receptor signaling in resident synoviocytes for development of full blown destructive arthritis

Chronic arthritis is characterized by persistent joint inflammation and concomitant joint destruction. IL-17 is a novel proinflammatory T-cell cytokine suspected to be involved in inflammatory and autoimmune diseases such as rheumatoid arthritis. Here, we report that IL-17 receptor (IL-17R) signaling is required in resident synovial cells for full progression of chronic synovitis and bone erosion. Repeated injections of Gram-positive bacterial cell wall fragments (SCW) directly into the knee joint of naive IL-17R-deficient (IL-17R-/-) mice had no effect on the acute phase of arthritis but prevented progression to chronic destructive synovitis as was noted in wild-type (wt) mice. Micro-array analysis revealed significant down-regulation of leukocyte-specific chemokines, selectins, collagenase-3, and IL-1 in the synovium of IL-17R-/- mice. Bone marrow (BM) chimeric mice revealed the need for IL-17/IL-17R signaling in resident synovial cells for development of full blown synovitis. Chimeric mice of host wt and donor IL-17R-/- BM cells developed destructive synovitis in this chronic relapsing SCW arthritis model similar to wt → wt chimeras. In contrast, chimeric mice of host IL-17R-/- and donor wt BM cells were protected from full blown destructive arthritis similar to IL-17R-/- → IL-17R-/- chimeras. These data strongly suggest T-cell IL-17-IL-17R signaling in resident synovial cells to be a pivotal mechanism through which an acute macrophage-driven joint inflammation progresses into a chronic destructive synovitis. Prevention of local synovial IL-17-IL-17R signaling warrants consideration as a therapeutic target in chronic destructive arthritis.

Effects of B7.2−/− Mature Dendritic Cells on Tolerance Induction to Alloantigens in Fetal Mice Following In Utero Transplantation with Lineage Depleted Bone Marrow.

In utero hematopoietic stem cell transplantation (IUT) has the potential to cure a variety of marrow stem cell defects without using marrow ablative therapy. However IUT for diseases other than SCID has been unsuccessful. To better understand the barriers to successful IUT we wanted to define the role of the B7.1/B7.2 co-stimulatory molecules in inducing tolerance to allogeneic donor bone marrow cells in the fetal murine recipient. We studied the relative role of B7.1 and B7.2 expression on dendritic cells (DC) on engraftment and in generating donor specific tolerance in fetal mice. Mature DC (mDC) from B7.1−/− or B7.2−/− donors and wild type (wt) lineage depleted (lin−) C57Bl/6 (B6) bone marrow (BM) were injected into gestational day (GD) 14 Balb/c fetuses. Recipients of lin− wt BM and B7.1−/− mDC had a significantly lower survival (47.4%, p<0.01) associated with mild-moderate GvHD compared to the recipients of B7.2−/− mDC and lin− BM (82.3%) where none developed GvHD. Engraftment results in blood at 6 weeks post IUT showed, B7.1−/− recipients had multilineage engraftment (4.7±0.8% T cells and 5.7± 1.1% granulocytes) in their blood, but by 12 weeks, only donor CD3+ (predominantly CD8+) cells (2.1±1.3%) were present. The percent H2Kb+ (donor) T cells (predominantly CD4+) in the blood of recipients of lin− wt BM and B7.2−/− was 11.8±8.5% at 6 weeks p<0.001 and 6.5±2.5% at 12 weeks, p=0.006. The circulating donor CD4+ cells were Th2 (CD4+CD25−IL4+IL10+) and Treg (CD4+CD25+IL4−IL10−). Both fractions inhibited the T cell proliferative response in the MLR. Long term engraftment in thymic tissues was found in the tolerant recipients of lin− wt BM and B7.2−/− mDC (13.4±8.3% donor CD3+ T cells). We also found prolonged (rejection by day 36) acceptance of donor skin grafts in 7 of 12 recipients of B7.2−/− mDC and 2 of 5 recipients of B7.2−/− mDC and lin−BM. All third party C3H grafts were rejected by day 14 and 80% of the Balb/c (self) skin grafts were permanently accepted. We hypothesized that tolerized animals would behave similarly to recipients of megadoses of syngeneic BM with an increase in multilineage engraftment. We injected a total of 200x106 male wt B6 lin− BM cells over 5 days into adult IUT recipients of B7.1−/− or B7.2−/− mDC ± lin− wt BM and wt age-matched allogeneic and syngeneic (female) controls. Mice that had received B7.2−/− mDC + lin− BM in utero showed multi-lineage engraftment in the blood. In contrast, the in utero recipients of B7.1−/− mDC + lin− BM showed no significant engraftment (p<0.05). In conclusion, donor DC costimulatory molecules significantly affect survival, engraftment and GvHD; and these responses to B7.2−/− mDC and lin− BM appear to be mediated by both Th2 and Treg donor cells.

FLT3 ITD and FLT3 Tyrosine Kinase Domain Mutants Induce Different Phenotypes in a Murine Bone Marrow Transplant Model.

Activating mutations of FLT3 are frequent in patients with acute myeloid leukemia (AML). Two distinct types of FLT3 mutations are most common: Internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence in approximately 30% of patients with AML and point mutations within the second tyrosine kinase domain (TKD) in about 7% of AML patients. Patients carrying the FLT3 ITD mutation seem to have a significantly worse prognosis, whereas the impact of TKD mutations on clinical outcome has not yet been determined. Recently, point mutations within the activation loop of FLT3 were also found in a significant percentage of infant and childhood acute lymhoblastic leukemia (ALL).Previous studies demonstrate that mice receiving transplants of bone marrow retrovirally infected with FLT3 ITD develop a myeloproliferative disease. The effect of FLT3 TKD mutations in vivo has not yet been investigated.To examine the transforming properties of FLT3 TKD mutants in primary hematopoietic cells, we used a bone marrow transplant model (BMT). Therefore we transduced bone marrow with retrovirus expressing either FLT3 D835Y or FLT3 I836M+R and transplanted it to lethally irradiated syngeneic recipient mice. As control we also transplanted mice with FLT3 WT and ITD infected bone marrow, respectively. We found that mice transplanted with FLT3 ITD developed a myeloproliferative disorder in mice, as previously described. In contrast, mice transplanted with FLT3 TKD mutants developed a lymphoid disease with distinct hematologic manifestation.Most recipients of FLT3 TKD transduced bone marrow developed T lymphoma syndrome, characterized by massive enlargement of thymus and lymph nodes. Some mice developed a B lymphoid leukemia with splenomegaly and enlarged lymph nodes. Interestingly, the disease latency of 53 to 183 days (median 102 days) of FLT3 TKD mutants contrasted with FLT3 ITD mice, which succumbed myeloproliferative disease within 53 to 70 days (median 58 days).The lymphoid manifestation and longer latency of FLT3 TKD in a murine BMT model together with the absent influence of FLT3 TKD mutations on clinical outcome of AML patients suggest differences in cell signaling between FLT3 TKD mutants and FLT3 ITD. The TKD mutants seem to require lymphoid cell context for full malignant transformation, whereas FLT3 ITDs transform myeloid cells.

Gadd45a and Gadd45b Protect Haematopoetic Cells from Ultraviolet Radiation Induced Apoptosis Via Distinct Signaling Pathways.

Gadd45 expression, which is stress inducible, has been associated with growth arrest, but the exact role of gadd45 family genes in apoptosis still remains unclear. We have found that myeloid progenitor cells from gadd45a and gadd45b-deficient mice are more sensitive to ultra-violet radiation, VP-16 or daunorubicin induced apoptosis. indicating that gadd45a or gadd45b protect haematopoetic cells from DNA damaging agents. To determine, how gadd45a or gadd45b proteins exert their anti-apoptotic function, bone marrow cells from wild-type and gadd45a or gadd45b deficient mice were exposed to ultraviolet radiation (UV) and analyzed for expression of stress responsive kinases, including JNK and p38. It was observed that P38 and JNK were activated in wt bone marrow cells in response to UV but not in bone marrow cells defecient in gadd45a. Also, the transcription factor NF-kB was activated in wt bone marrow cells, but not in gadd45a−/− cells. The pharmacological inhibitor SB203580 specific for p38, increased apoptosis in reponse to UV, indicating that p38 is implicated in signaling myeloid cell survival. SB203580 was observed also to inhibit the expression of certain NF-kB target genes, including cIAP-1, c-IAP-2, bcl-2 and bcl-xl, in gadd45a+/+ cells but not in gadd45a deficient bone marrow cells. Taken together this data provides first evidence for the role gadd45a plays in the control of hematopoietic cell survival in response to UV, via modulation of P38 MAPK and NF-kB signaling pathways. Unlike in gadd45a−/− bone marrow cells, p38 activation appeared not to be impaired in gadd45b−/− cells, indicating that gadd45b is not involved in p38 activation in myeloid cells. However, UV induced JNK activation was sustained in gadd45b−/− myeloid cells compared to wt cells, indicating that gadd45b is a negative modulator of UV induced JNK signaling in myeloid cells. UV induced activation of MKK4 an upstream regulator of JNK also was impaired in gadd45b−/−. NF-kB was also found activated in wt cells, but not in gadd45b−/− cells. This data indicates that in bone marrow cells exposed to UV, NF-kB induced expression of Gadd45b plays a protective role against UV induced apoptosis via inhibition of MKK4 kinase which in turn results in suppression of JNK activity. Taken together this data provides evidence that Gadd45a and Gadd45b protect haematopoetic cells from genotoxic-stress induced apoptosis via distinct signaling pathways.

Donor Antigen Presenting Cell CD80/86 Function Is Required for Eliciting T Cell Resistance Against Progenitor Cell Engraftment in an MHC Matched Allogeneic Non-Myeloablative Transplantation Model.

Immune mediated resistance is a concern in allogeneic bone marrow transplantation (BMT) particularly in the setting of nonmyeloablative conditioning regimens or in situations where prior sensitization to hematopoietic alloantigens has occurred (ie aplastic anemia). This resistance can lead to both delayed reconstitution and rejection. In this investigation, two models of T cell mediated allogeneic bone marrow graft rejection were employed: 1) an MHC disparate, donor antigen sensitized model, ie BALB/c (H-2d) recipients sensitized to B6 (H-2b) hematopoietic antigens transplanted with a high dose (1x107) of B6 BM-TCD following ablative (9.0Gy TBI) conditioning. Resistance in this model was assessed by the presence of donor CFU progenitors early (Day 5) post transplant and 2) a non-myeloablative mHag disparate model, ie C57BL/6 TCD BM à C3H.SW recipients conditioned with 5.5 Gy TBI. In this latter model, BM from B6 wild-type donors at doses <1x107 cells leads to transient donor peripheral chimerism (assessed by monitoring Ly9.1 expression) peaking at day 7–10 post transplant. To study the role of donor APC function in the transplant innoculum in both models, bone marrow was utilized from donors deficient in CD80 and CD86, yielding a “crippled” APC population incapable of co-stimulation through these two primary pathways. In the antigen sensitized model, (ie Tmemory mediated) both lineage committed (CFU-IL3) and multipotential (CFU-HPP) progenitor activity was absent from recipients of both wild-type or CD80/86 −/− BMC. This data suggests that CD80/86 mediated co-stimulation from direct recognition of donor APC is dispensible for eliciting resistance when memory T cells are involved. However, in the unsensitized model, despite the potential for indirect donor antigen presentation, recipients of CD80/86−/− bone marrow exhibited significant engraftment by one month post-transplant (in contrast, recipients of wt bone marrow had universally these rejected grafts by this time point). Significant chimerism in recipients of CD80/86−/− BM was detectable in the B as well as T lymphocyte compartments (>50%). Consistent with the importance of the presence of these co-stimulatory pathways on the donor APC population, 5/7 recipients of mixed (1:1) BM (CD80/86−/− + B6-wt) transplants rejected their grafts. These findings suggest that removal of CD80/86 dependent co-stimulatory pathways in the donor APC population may be insufficient for promoting engraftment in cases where there has been prior donor antigen sensitization, i.e. in the presence of host Tmemory populations. However, absence of CD80/86 co-stimulatory pathways in the donor APC population may be sufficient for overcoming resistance in an MHC matched allo-transplant setting in which prior donor antigen sensitization has not occurred, ie in the presence of host Tnaive populations. These findings have implications in the development of improved non-myeloablative conditioning protocols to facilitate engraftment without global immunosuppressive conditioning.

C-Kit Receptor is Essential for Functional Cardiac Repair Post-myocardial Infarction

Recent findings report on the potential for bone marrow (BM)-derived cell therapy post-myocardial infarction (MI). With ischemic injury, various cytokine signaling mechanisms are activated. This leads to cell mobilization and homing to the site of injury. Stem cell factor (SCF) and its receptor c-kit have been particularly implicated in this mechanism. We sought to directly elucidate the therapeutic contribution of c-kit post-MI, by comparing functional repair in c-kit deficient mice compared to littermate controls (WT). Methods: MI was created by ligation of the left anterior descending coronary artery in heterozygous c-kit deficient W/Wv and WT mice. To elucidate the possibility of phenotype rescue, a third group of c-kit deficient W/Wv mice were transplanted with lentivirally gene transduced (enGFP) WT BM (BMT). Three months were allowed for BM repopulation prior to surgery. Five weeks post-MI, in vivo LV pressure-volume performance and pathology was evaluated. Results: There was worsening of heart function and greater cardiac dilatation post-MI in c-kit deficient mice compared to WT. The LV end-systolic volume (VES; relative volume units (RVU)) increased significantly in c-kit deficient mice compared to controls (26.60+/−0.63 vs. 22.55+/−0.62 RVU in WT, p<0.0001), and heart/body weight ratios followed similarly compared to controls (0.01315+/−0.0008 vs. 0.0090+/−0.0008 in WT, p<0.0001). In contrast, the LV end-systolic pressure (PES) was significantly worsened in c-kit deficient mice (75.75+/−1.40 vs. 85.60+/−1.39 mmHg in WT, p<0.0001). On the other hand, the chimeric c-kit deficient mice transplanted with gene-marked WT BM demonstrated a significant reduction in LV VES and heart/body weight ratios (22.37+/−0.57 RVU and 0.0074+/−0.0005, p<0.0001 for both, compared to c-kit MI, non-chimeric group), and improvement in LV PES (81.94+/−1.37, p=0.0052). There was no significant difference when compared to the WT group. Assessment of the existence of differentiated enGFP cells in the heart is presently underway using two photon microscopy. Conclusion: We have demonstrated the importance of c-kit receptor signaling for preventing the deterioration of cardiac function and cardiac hypertrophy post-MI. Further illustrating its importance for future therapeutic studies, we have also shown that rescue of c-kit deficiency can be achieved by WT gene-marked BMT. c-kit function thus appears to play a crucial protective role in remodeling post-MI.

179. Oncoretroviral Vector-Mediated Gene Therapy for Murine WAS Fails To Correct Most of the Deficient Biological Responses to S. pneumonia or M. bovis

The Wiskott-Aldrich syndrome is an X-linked congenital condition manifesting as variable degrees of immunodeficiency and thrombocytopenia. Bone marrow transplantation is apparently curative, but frequent lack of donor availability, and variable outcomes after transplantation with matched unrelated donors, indicate a need for alternative therapies. A murine model of WAS demonstrates features similar to the clinical condition, including lymphocytopenia, thrombocytopenia, and increased susceptibility to certain infectious agents. WAS- mice show improvement in several cellular and physiological parameters after transplantation with WT bone marrow cells. Gene corrected animals, generated by retroviral-vector mediated transduction of murine bone marrow cells followed by transplantation into lethally irradiated recipients, show improvement in the colitis seen in a subset of these animals, and also demonstrate improvement in their deficient secondary T-cell response to influenza inoculation. Here we demonstrate that WAS- mice show increased susceptibility to, and mortality from, pneumonia following intranasal inoculation with S. pneumonia. Specifically, after intranasal inoculation with 105 cfu of luciferase-labeled S. pneumonia, 0 of 10 WT WAS (C57Bl/6, 5th generation backcross from 129SvEv) mice developed pneumonia or died, while 6 of 10 WAS- mice developed pneumonia and died. However, gene-corrected animals did not show a significant improvement in these outcomes. After inoculation with a lower dose (0.5 × 105 cfu) of S. pneumonia, 4 of 7 gene corrected mice, 5 of 6 negative control mice (WAS- mice transplanted with WAS-, GFP-vector transduced bone marrow cells), and 4 of 8 positive controls (WAS- mice transplanted with WT, GFP-vector transduced bone marrow cells) developed pneumonia and died. Susceptibility did not correlate with the extent of engraftment of gene corrected cells in any hematologic lineage. We conclude that diminished resistance of positive control mice was at least partially responsible for the lack of observable phenotypic correction. We have also observed reduced clearance of M. bovis (bcg) from the organs of intravenously inoculated WAS- animals. Here too, however, gene-corrected animals showed only inconsistent indications of improvement in M. bovis clearance. Specifically, gene corrected WAS- mice showed improved clearance from liver, but no overall improvement in clearance from spleen or lung. Clearance correlated with the extent of engraftment of gene corrected lymphocytes and monocytes in liver and spleen, but not in lung. Our results suggest that technical improvements will be required in order to evaluate the biological efficacy of gene therapy for murine WAS. Specifically, we are investigating whether lethal irradiation alone, lack of a perfect genetic match among donors and recipients, or GFP expression diminishes resistance to these agents in recipients of control vector transduced, WT bone marrow cells, as well as whether improved vector design could enhance efficacy.

Mobilization studies in mice deficient in either C3 or C3a receptor (C3aR) reveal a novel role for complement in retention of hematopoietic stem/progenitor cells in bone marrow

AbstractThe mechanisms regulating the homing/mobilization of hematopoietic stem/progenitor cells (HSPCs) are not fully understood. In our previous studies we showed that the complement C3 activation peptide, C3a, sensitizes responses of HSPCs to stromal-derived factor 1 (SDF-1). In this study, mobilization was induced with granulocyte colony-stimulating factor (G-CSF) in both C3-deficient (C3–/–) and C3a receptor–deficient (C3aR–/–) mice as well as in wild-type (wt) mice in the presence or absence of a C3aR antagonist, SB 290157. The data indicated (1) significantly increased G-CSF–induced mobilization in C3–/– and C3aR–/– mice compared with wt mice, (2) significantly accelerated and enhanced G-CSF–induced mobilization in wt, but not in C3–/– or C3aR–/–, mice treated with SB 290157, and (3) deposition of C3b/iC3b fragments onto the viable bone marrow (BM) cells of G-CSF–treated animals. Furthermore, mobilization studies performed in chimeric mice revealed that wt mice reconstituted with C3aR–/– BM cells, but not C3aR–/– mice reconstituted with wt BM cells, are more sensitive to G-CSF–induced mobilization, suggesting that C3aR deficiency on graft-derived cells is responsible for this increased mobilization. Hence we suggest that C3 is activated in mobilized BM into C3a and C3b, and that the C3a-C3aR axis plays an important and novel role in retention of HSPCs (by counteracting mobilization) by increasing their responsiveness to SDF-1, the concentration of which is reduced in BM during mobilization. The C3a-C3aR axis may prevent an uncontrolled release of HSPCs into peripheral blood. These data further suggest that the C3aR antagonist SB 290157 could be developed as a drug to mobilize HSPCs for transplantation.

Tolerance induction by intrathymic expression of P0.

Genetic deficiency or instability of myelin protein zero (P0) results in hereditary motor sensory neuropathy. In view of recent advances in gene therapy, substitution of the molecular defect may become realistic in the near future. Here we investigate the impact of genetic deficiency of P0 on selection of the autoreactive T cell repertoire in the corresponding mouse model. We show that P0 mRNA transcripts are expressed in thymic stroma, similar to other myelin proteins and that expression of intact P0 protein can be detected by Western blot. Using a library of overlapping 20mer peptides spanning the entire length of P0 and applying the ELISPOT technique, we detected a strong immune response toward P0 extracellular domain peptide aa 41-60 in P0(-/-) knockout mice, but not in heterozygous P0(+/-) or wild-type (wt) mice. In addition, one cryptic epitope and two subdominant epitopes of P0 were identified. Using P0(-/-) into wt bone marrow (BM) chimeras we found that P0 expression in the host suffices for full tolerance induction, which is in line with its presence in thymic stroma. However, repopulation of P0(-/-) mice with wt BM led to partial induction of tolerance, suggesting that BM derived cells can also express this protein. Our findings may have implications for secondary autoimmunity developing after gene therapy in hereditary neuropathies and other diseases with genetically determined protein deficiency, because the repaired protein will then represent a foreign, nontolerized Ag.

Requirement of local synovial IL-17 receptor signaling in the progression of chronic synovitis and bone erosion in arthritis

IL-17 is a proinflammatory cytokine that is suspected to be involved in development of rheumatoid arthritis (RA). However, the role of IL-17–IL-17 receptor signaling in the effector phase of arthritis, including requirement of this signaling pathway in synoviocytes, is still not fully elucidated. Here we demonstrate, using IL-17 receptor deficient (IL-17R-/-) mice, a requirement for IL-17R signaling in immune reactivity and progression of synovitis and bone erosion in a T cell mediated mBSA arthritis model and chronic streptococcal cell wall arthritis. Of great interest, chimeric mice of host wild-type (wt) and donor IL-17R-/- bone marrow (BM) cells developed chronic synovitis similar to that in wt/wt chimeras. In contrast, chimeric mice of host IL-17R-/- and donor wt BM cells were protected from progressive synovitis similar to IL-17R-/-/ IL-17R-/- chimeras, suggesting a crucial role of resident synovial cells. Weakened mobilization of neutrophils into the joint and downregulation of synovial mRNA expression for leukocyte specific chemokines and selectins, and MMP-1 was found in IL-17R-/- mice. These data suggest that, in addition to the role played by IL-17–IL-17R signaling in development of immunity, synovial IL-17–IL-17R signaling plays a critical role in the effector phase of arthritis. Activation of IL-17R on fibroblast-like synoviocytes appears pivotal.

Bone marrow transplantation reveals an essential synergy between neuronal and hemopoietic cell neurokinin production in pulmonary inflammation

Neurogenic inflammation is believed to originate with the antidromic release of substance P, and of other neurokinins encoded by the preprotachykinin A (PPT-A) gene, from unmyelinated nerve fibers (C-fibers) following noxious stimuli. Consistent with this concept, we show here that selective sensory-fiber denervation with capsaicin and targeted deletion of the PPT-A gene protect murine lungs against both immune complex–mediated and stretch-mediated injuries. Reconstitution of PPT-A gene–deleted mice with WT bone marrow does not abrogate this protection, demonstrating a critical role for PPT-A gene expression by sensory neurons in pulmonary inflammation. Surprisingly, reconstitution of WT mice with PPT-A gene–deficient bone marrow also confers protection against pulmonary injury, revealing that PPT-A gene expression in hemopoietic cells has a previously unanticipated essential role in tissue injury. Taken together, these findings demonstrate a critical synergy between capsaicin-sensitive sensory fibers and hemopoietic cells in neurokinin-mediated inflammation and suggest that such synergy may be the basis for a stereotypical mechanism of response to injury in the respiratory tract.

Bone marrow transplantation reveals an essential synergy between neuronal and hemopoietic cell neurokinin production in pulmonary inflammation.

Neurogenic inflammation is believed to originate with the antidromic release of substance P, and of other neurokinins encoded by the preprotachykinin A (PPT-A) gene, from unmyelinated nerve fibers (C-fibers) following noxious stimuli. Consistent with this concept, we show here that selective sensory-fiber denervation with capsaicin and targeted deletion of the PPT-A gene protect murine lungs against both immune complex-mediated and stretch-mediated injuries. Reconstitution of PPT-A gene-deleted mice with WT bone marrow does not abrogate this protection, demonstrating a critical role for PPT-A gene expression by sensory neurons in pulmonary inflammation. Surprisingly, reconstitution of WT mice with PPT-A gene-deficient bone marrow also confers protection against pulmonary injury, revealing that PPT-A gene expression in hemopoietic cells has a previously unanticipated essential role in tissue injury. Taken together, these findings demonstrate a critical synergy between capsaicin-sensitive sensory fibers and hemopoietic cells in neurokinin-mediated inflammation and suggest that such synergy may be the basis for a stereotypical mechanism of response to injury in the respiratory tract.

Distinct contributions of TNF and LT cytokines to the development of dendritic cells in vitro and their recruitment in vivo

TNF/LTalpha/LTbeta (tumor necrosis factor/lymphotoxin-alpha/lymphotoxin-beta) triple knockout (KO) mice show a significant reduction of dendritic cell (DC) number in the spleen, presumably due to defective recruitment and/or production. To distinguish between these possibilities, DCs were generated from bone marrow (BM) cultures prepared from wild-type (wt) and mutant mice in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The yield of CD11c(+) major histocompatibility complex (MHC) class II(+) DCs generated from TNF/LTalpha/LTbeta(-/-) BM culture was significantly reduced compared with wt BM culture. In order to further dissect the individual pathways responsible for defective DC properties observed in TNF/LTalpha/LTbeta(-/-) mice, the panel of TNF/LT ligand and receptor single KO mice were used. The production of DCs from BM culture was significantly reduced in TNF(-/-) and TNF receptor (TNFR) p55(-/-) mice, but normal in LTalpha(-/-), LTbeta(-/-), LTbetaR(-/-) mice. Recombinant TNF (rTNF) exogenously added to TNF/LTalpha/LTbeta(-/-) BM cultures could reverse this defect, and blocking antibodies showed partial effect on BM cultures of wt mice. Conversely, numbers of mature DCs in spleen were significantly decreased in LTalpha(-/-), LTbeta(-/-), LTbetaR(-/-) mice, but not in TNF(-/-) and TNFRp55(-/-) mice. These results reveal 2 distinct contributions of TNF/LT cytokines. First, TNF acting through TNF receptor is involved in the development/maturation of DCs in BM progenitor cultures, but this function appears to be redundant in vivo. Second, the microenvironment in peripheral lymphoid organs associated with LTalpha/LTbeta-LTbetaR signaling and chemokine production is critical for recruitment efficiency of DCs, and this pathway is indispensable.

Targeted gene correction in the mdx mouse using short DNA fragments: towards application with bone marrow-derived cells for autologous remodeling of dystrophic muscle.

In muscle, mutant genes can be targeted and corrected directly by intramuscular (i.m.) injection of corrective DNA, or by ex vivo delivery of DNA to myogenic cells, followed by cell transplantation. Short fragment homologous replacement (SFHR) has been used to repair the exon 23 nonsense transition at the Xp21.1 dys locus in cultured cells and also, directly in tibialis anterior from male mdx mice. Whilst mdx dys locus correction can be achieved in up to 20% of cells in culture, much lower efficiency is evident by i.m. injection. The major consideration for application of targeted gene correction to muscle is delivery throughout relevant tissues. Systemically injected bone marrow (BM)-derived cells from wt C57BL/10 ScSn mice are known to remodel mdx muscle when injected into the systemic route. Provided that non muscle-derived cell types most capable of muscle remodeling activity can be more specifically identified, isolated and expanded, cell therapy seems presently the most favorable vehicle by which to deliver gene correction throughout muscle tissues. Using wt bone marrow as a model, this study investigates systemic application of bone marrow-derived cells as potential vehicles to deliver corrected (ie wt) dys locus to dystrophic muscle. Intravenous (i.v.) and intraperitoneal (i.p.) injections of wt BM were given to lethally and sub-lethally irradiated mdx mice. Despite both i.v. and surviving i.p. groups containing wt dys loci in 100% and less than 1% of peripheral blood nuclei, respectively, both groups displayed equivalent levels of wt dys transcript in muscle RNA. These results suggest that the muscle remodeling activity observed in systemically injected BM cells is not likely to be found in the hemopoietic fraction.

IKKβ Is Essential for Protecting T Cells from TNFα-Induced Apoptosis

Transcription factor NF-κB, whose activation depends on the IKKβ catalytic subunit of the IκB kinase, was assigned with both anti- and proapoptotic functions in T lymphocytes. To critically evaluate these functions, we transferred Ikkβ −/− or wild-type (wt) fetal liver (FL) stem cells into lethally irradiated mice. Ikkβ −/− radiation chimeras show thymic rudiments, aberrant lymphoid organs, and absence of T cells. T lymphopoiesis is rescued when Ikkβ −/− stem cells are cotransferred with wt bone marrow, suggesting that IKKβ may mediate its lymphopoietic function via extrinsic factors. However, almost normal development of Ikkβ −/− T cells is observed upon removal of type 1 TNFα receptor, indicating that TNFα signaling accounts for the absence of Ikkβ −/− T cells. Indeed, Ikkβ −/− radiation chimeras exibit elevated circulating TNFα, and Ikkβ −/− thymocytes display increased TNFα sensitivity.

Consequences of Fas-ligand and perforin expression by colon T cells in a mouse model of inflammatory bowel disease

Background & Aims: We describe a type of colitis that develops after transplantation of nonallogeneic wt bone marrow cells into T cell– and natural killer cell–deficient Tgϵ26 mice (BM→Tgϵ26). In these animals, severe wasting and inflammation of the colon correlates with the expansion of mucosal T lymphocytes that displays cytotoxic activity. The aims of this study were to determine the relative contribution of perforin and Fas ligand (Fas-L) expression to the cytotoxic action of these T cells and to examine the influence of each pathway in this model of colitis. Methods: Colonic T cells were tested for their ability to mediate Fas- and perforin-dependent killing in redirected cytotoxicity assays. Bone marrow cells from donor mice lacking either Fas-L ( gld mice) or perforin (PFP null mice) or both molecules were used to reconstitute Tgϵ26 mice. Results: Colon cytotoxic T lymphocyte displayed both Fas- and perforin-dependent killing. Deficiency in perforin, but not Fas-L, resulted in reduced incidence of wasting and, to a lesser extent, severe colitis in BM→Tgϵ26 animals. Conclusions: Colon T cells from BM→Tgϵ26 mice express both perforin and Fas-L. Although neither pathway is critical in the development of colitis, perforin does have a measurable influence on disease in the BM→Tgϵ26 colitis model. GASTROENTEROLOGY 1998;115:849-855

Apoptosis with FasL+ cell infiltration in the periphery and thymus of corrected autoimmune mice.

Fas (CD95) ligand (L) is a death factor that binds to its receptor, Fas, and induces apoptotic cell death, a crucial process in immunological tolerance. gld (generalized lymphoproliferative disorder) mice, which have a point mutation in the FasL gene, develop spontaneous systemic autoimmune syndromes characterized by hypergammaglobulinaemia and lymphoid hyperplasia owing to accumulation of abnormal B220+ CD3+ cells. Transplantation of wild-type (wt) bone marrow cells into old gld mice on the same strain background results in normalization of autoimmune syndromes. We characterized the cellular mechanisms (functionally and histologically) of the above phenomena in gld mice after bone marrow transplantation (BMT) to determine the role of apoptosis via Fas/FasL interactions in inducing and maintaining self-tolerance in vivo. Activated splenocytes from wt and BMT (wt to gld) mice showed significant cytotoxic activity against Fas transfectant cells while those from BMT (gld to gld) mice did not. Cells in the thymus, spleen and lymph nodes of gld mice uniformly upregulated Fas expression and were sensitive to Fas-mediated apoptosis compared with those in wt mice. Cells sensitive to Fas-mediated apoptosis in gld mice resided not only among abnormal B220+ CD3+ cells but also among conventional lymphocytes. More importantly, histological analysis revealed that cells in the spleen, lymph nodes and thymus frequently underwent apoptosis with infiltration of FasL+ cells in BMT (wt to gld) mice compared with BMT (gld to gld) mice. Our results indicated that apoptosis via Fas/FasL interactions can directly eliminate pathogenic cells responsible for autoimmunity in the periphery and possibly in the thymus in vivo.