Immune-mediated bone marrow failure in C57BL/6 mice
2014; Elsevier BV; Volume: 43; Issue: 4 Linguagem: Inglês
10.1016/j.exphem.2014.12.006
ISSN1873-2399
AutoresJichun Chen, Marie J. Desierto, Xingmin Feng, Angélique Biancotto, Neal S. Young,
Tópico(s)Immune Response and Inflammation
Resumo•We developed a new model of immune-mediated bone marrow failure in C57BL/6 mice•Irradiation dose and specific donor-recipient combination are critical to model development•Bone marrow of model animals displayed clonal T-cell expansion and T-cell Fas ligand upregulation•Inflammatory cytokine interferon γ and tumor necrosis factor α were increased•Mice carrying germline Fas deletion had bone marrow destruction significantly attenuated We established a model of immune-mediated bone marrow (BM) failure in C57BL/6 (B6) mice with 6.5 G total-body irradiation followed by the infusion of 4-10 × 106 lymph node (LN) cells/recipient from Friend leukemia virus B/N (FVB) donors. Forty-three percent of animals succumbed, with surviving animals showing marked declines in blood neutrophils, red blood cells, platelets and total BM cells at 8 to 14 days following LN cell infusion. Lowering the total-body irradiation dose to 5 G or altering the LN source from FVB to BALB/cBy donors failed to produce BM destruction. Affected animals showed significant expansion and activation of CD8 T lymphocytes in both the blood and BM; cytotoxic T cells had elevated Fas ligand expression and were oligoclonal, mainly displaying Vβ7 and Vβ17 T cell receptors. There were significant increases in blood plasma interferon γ and tissue necrosis factor α in affected animals. Chemokine ligands CCL3, CCL4, CCL5, CCL20, CXCL2, and CXCL5 and hematopoietic growth factors G-CSF, M-CSF, GM-CSF, VEGF were also elevated. In B6 mice carrying a Fas gene mutation, BM failure was attenuated when they were infused with FVB LN cells. Our model establishes a useful platform to define the roles of individual genes and their products in immune-mediated BM failure. We established a model of immune-mediated bone marrow (BM) failure in C57BL/6 (B6) mice with 6.5 G total-body irradiation followed by the infusion of 4-10 × 106 lymph node (LN) cells/recipient from Friend leukemia virus B/N (FVB) donors. Forty-three percent of animals succumbed, with surviving animals showing marked declines in blood neutrophils, red blood cells, platelets and total BM cells at 8 to 14 days following LN cell infusion. Lowering the total-body irradiation dose to 5 G or altering the LN source from FVB to BALB/cBy donors failed to produce BM destruction. Affected animals showed significant expansion and activation of CD8 T lymphocytes in both the blood and BM; cytotoxic T cells had elevated Fas ligand expression and were oligoclonal, mainly displaying Vβ7 and Vβ17 T cell receptors. There were significant increases in blood plasma interferon γ and tissue necrosis factor α in affected animals. Chemokine ligands CCL3, CCL4, CCL5, CCL20, CXCL2, and CXCL5 and hematopoietic growth factors G-CSF, M-CSF, GM-CSF, VEGF were also elevated. In B6 mice carrying a Fas gene mutation, BM failure was attenuated when they were infused with FVB LN cells. Our model establishes a useful platform to define the roles of individual genes and their products in immune-mediated BM failure. Aplastic anemia (AA), the paradigm of bone marrow (BM) failure syndromes, is anemia, neutropenia, and thrombocytopenia, with a hypocellular BM [1Scheinberg P. Young N.S. How I treat acquired aplastic anemia.Blood. 2012; 120: 1185-1196Crossref PubMed Scopus (293) Google Scholar]. Although the etiology is unclear, most AA patients respond to immunosuppressive therapy [2Marsh J.C. Bacigalupo A. 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Retreatment with rabbit anti-thymocyte globulin and ciclosporin for patients with relapsed or refractory severe aplastic anaemia.Br J Haematol. 2006; 133: 622-627Crossref PubMed Scopus (134) Google Scholar], implicating a pathophysiologic destruction of hematopoietic stem cells (HSCs) and progenitors by the immune system [6Kordasti S. Marsh J. Al-Khan S. et al.Functional characterization of CD4+ T cells in aplastic anemia.Blood. 2012; 119: 2033-2043Crossref PubMed Scopus (114) Google Scholar]. The immune mechanism was also supported by laboratory observations of Th1 immune response cytokine interferon γ (IFN-γ) [7Selleri C. Maciejewski J.P. Sato T. Young N.S. Interferon-gamma constitutively expressed in the stromal microenvironment of human marrow cultures mediates potent hematopoietic inhibition.Blood. 1996; 87: 4149-4157Crossref PubMed Google Scholar, 8Selleri C. Sato T. Anderson S. Young N.S. Maciejewski J.P. 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Solomou E.E. et al.Rabbit ATG but not horse ATG promotes expansion of functional CD4+CD25highFOXP3+ regulatory T cells in vitro.Blood. 2008; 111: 3675-3683Crossref PubMed Scopus (186) Google Scholar].Bone marrow failure has been successfully modeled in animals by the infusion of allogeneic lymph node (LN) cells from donors mismatched at major histocompatibility complex (MHC) or minor-histocompatibility (minor-H) antigens [12Barnes D.W. Mole R.H. Aplastic anaemia in sublethally irradiated mice given allogeneic lymph node cells.Br J Haematol. 1967; 13: 482-491Crossref PubMed Scopus (13) Google Scholar, 13Chen J. Animal models for acquired bone marrow failure syndromes.Clin Med Res. 2005; 3: 102-108Crossref PubMed Scopus (50) Google Scholar]. Barnes and Mole produced the first mouse model of immune-mediated AA by infusing 1-10 × 106 LN cells from C3H donors into CBA/H recipients preirradiated at 450–600 rads of total-body irradiation (TBI). Fatal AA developed in recipient animals, which had reduced blood cell counts and an empty BM. Allogeneic LN cells were responsible for the pathology, since TBI alone or TBI plus infusion of irradiation-inactivated LN cells were ineffective in producing BM damage [12Barnes D.W. Mole R.H. Aplastic anaemia in sublethally irradiated mice given allogeneic lymph node cells.Br J Haematol. 1967; 13: 482-491Crossref PubMed Scopus (13) Google Scholar]. This pioneering work was extended to other strain combinations in different experimental settings to successfully recapitulate the major pathophysiologic features of BM failure and to enable the study of disease mechanisms and testing of therapeutic interventions [14Knospe W.H. Steinberg D. Speck B. Experimental immunologically mediated aplastic anemia (AA) in H-2k identical, Mls (M) locus different mice.Exp Hematol. 1983; 11: 542-552PubMed Google Scholar, 15Knospe W.H. Steinberg D. Gratwohl A. Speck B. Experimental immunologically mediated aplastic anemia (AA) in mice: cyclosporin A fails to protect against AA.Int J Cell Cloning. 1984; 2: 263-271Crossref PubMed Scopus (6) Google Scholar, 16Knospe W.H. Husseini S.G. Chiu K.M. Fried W. Immunologically mediated aplastic anemia in mice: evidence of hematopoietic stromal injury and injury to hematopoietic stem cells.Exp Hematol. 1994; 22: 573-581PubMed Google Scholar, 17Kubota K. Mizoguchi H. Miura Y. Kano S. Takaku F. Experimental hypoplastic marrow failure in the mouse.Exp Hematol. 1978; 6: 791-800PubMed Google Scholar, 18Nemoto K. Hayashi M. Abe F. et al.Therapy of experimental immunologically mediated aplastic anemia in mice by various immunosuppressive and antitumor agents.Transplant Proc. 1988; 20: 545-548PubMed Google Scholar].We produced two mouse models using TBI plus allogeneic LN cell infusion approaches [19Bloom M.L. Wolk A.G. Simon-Stoos K.L. Bard J.S. Chen J. Young N.S. A mouse model of lymphocyte infusion-induced bone marrow failure.Exp Hematol. 2004; 32: 1163-1172Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 20Chen J. Lipovsky K. Ellison F.M. Calado R.T. Young N.S. Bystander destruction of hematopoietic progenitor and stem cells in a mouse model of infusion-induced bone marrow failure.Blood. 2004; 104: 1671-1678Crossref PubMed Scopus (60) Google Scholar, 21Chen J. Ellison F.M. Eckhaus M.A. et al.Minor antigen h60-mediated aplastic anemia is ameliorated by immunosuppression and the infusion of regulatory T cells.J Immunol. 2007; 178: 4159-4168Crossref PubMed Scopus (60) Google Scholar]. First, MHC heterozygous hybrid B6D2F1 and CByB6F1 mice carrying H2b/d were given 5 G TBI and an infusion of 5 × 106 LN cells from parental C57BL/6 (B6) donors (H2b/b). Pancytopenia and marrow hypoplasia developed within 2 to 3 weeks, with pathologic features mimicking human AA [19Bloom M.L. Wolk A.G. Simon-Stoos K.L. Bard J.S. Chen J. Young N.S. A mouse model of lymphocyte infusion-induced bone marrow failure.Exp Hematol. 2004; 32: 1163-1172Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]. We then tested TBI plus B6 LN cell infusion into MHC-matched (H2b/b), minor-H mismatched, C.B10 recipients, and this specific strain combination also produced fatal BM failure [21Chen J. Ellison F.M. Eckhaus M.A. et al.Minor antigen h60-mediated aplastic anemia is ameliorated by immunosuppression and the infusion of regulatory T cells.J Immunol. 2007; 178: 4159-4168Crossref PubMed Scopus (60) Google Scholar]. In these models, BM destruction was mediated by expanded and activated donor T lymphocytes that targeted host BM cells [20Chen J. Lipovsky K. Ellison F.M. Calado R.T. Young N.S. Bystander destruction of hematopoietic progenitor and stem cells in a mouse model of infusion-induced bone marrow failure.Blood. 2004; 104: 1671-1678Crossref PubMed Scopus (60) Google Scholar]. Fas- and Fas ligand (FasL)-associated cell death was the major pathway responsible for elimination of HSCs, hematopoietic progenitors, and other BM cellular components [22Omokaro S.O. Desierto M.J. Eckhaus M.A. Ellison F.M. Chen J. Young N.S. Lymphocytes with aberrant expression of Fas or Fas ligand attenuate immune bone marrow failure in a mouse model.J Immunol. 2009; 182: 3414-3422Crossref PubMed Scopus (28) Google Scholar]; the perforin-granzyme B pathway played a minor role [23Sarcon A.K. Desierto M.J. Zhou W. et al.Role of perforin-mediated cell apoptosis in murine models of infusion-induced bone marrow failure.Exp Hematol. 2009; 37: 477-486Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar]. Although a Th17 response was active early [24de Latour R.P. Visconte V. Takaku T. et al.Th17 immune responses contribute to the pathophysiology of aplastic anemia.Blood. 2010; 116: 4175-4184Crossref PubMed Scopus (121) Google Scholar], Th1 cells were most important in mediating massive BM destruction [25Solomou E.E. Keyvanfar K. Young N.S. T-bet, a Th1 transcription factor, is up-regulated in T cells from patients with aplastic anemia.Blood. 2006; 107: 3983-3991Crossref PubMed Scopus (109) Google Scholar, 26Tang Y. Desierto M.J. Chen J. Young N.S. The role of the Th1 transcription factor T-bet in a mouse model of immune-mediated bone-marrow failure.Blood. 2010; 115: 541-548Crossref PubMed Scopus (27) Google Scholar]. Recent reports from others utilizing this model have provided new evidence of modulation of T-bet expression by Notch1 and Ezh2 expression and the functional role of regulatory Th1 immune responses [27Roderick J.E. Gonzalez-Perez G. Kuksin C.A. et al.Therapeutic targeting of NOTCH signaling ameliorates immune-mediated bone marrow failure of aplastic anemia.J Exp Med. 2013; 210: 1311-1329Crossref PubMed Scopus (57) Google Scholar, 28Tong Q. He S. Xie F. et al.Ezh2 Regulates Transcriptional and Posttranslational Expression of T-bet and Promotes Th1 Cell Responses Mediating Aplastic Anemia in Mice.J Immunol. 2014; 192: 5012-5022Crossref PubMed Scopus (55) Google Scholar].In the current work, we sought to model immune-mediated BM failure in B6 mice, as B6 are widely used in biomedical research, especially for the development of transgenic and knockout animals. Our goal was to establish an experimental platform to test the roles of individual genes and molecules in immune-mediated marrow destruction. We successfully induced BM failure in B6 mice with 6.5–7.0 G TBI plus the infusion of 4-10 × 106 LN cells from Friend leukemia virus B/N (FVB) donors. Recipient B6 mice developed severe pancytopenia and marrow hypocellularity. Oligoclonal expansion and activation of donor lymphocytes was characteristic. Affected animals also showed elevations in plasma inflammatory cytokines, chemokine ligands, and hematopoietic growth factors typical of marrow failure. We tested the utility of the model in mice deficient in Fas gene expression and found that BM failure was significantly attenuated, consistent with current understanding of the pathophysiology of BM failure.Materials and methodsAnimals and induction of bone marrow failureInbred B6, BALB/cBy (BALB) and FVB/NJ (FVB) mice, as well as induced mutants C57BL/6-Prf1tm1Sdz/J (Pfr−/−) and B6.MRL-Faslpr/J (Fas−/−) mice, were all obtained from the Jackson Laboratory (Bar Harbor, ME), and were bred and maintained in National Institutes of Health animal facilities under standard care and nutrition. Young adult male and female mice were used at 2–10 months of age. All animal studies were approved by the Institutional Animal Care and Use Committee at the National Heart, Lung, and Blood Institute.Inguinal, axillary, and lateral axillary LNs were collected from FVB or BALB donors, homogenized with a mini–tissue grinder (A. Daigger & Company, Vernon Hills, IL) in Iscove's Modified Dulbecco's Medium (Life Technologies Corporation, Grand Island, NY), washed, centrifuged, filtered through 90 μmol/L nylon mesh (Small Parts, Miami Lake, FL), and counted by a Vi-Cell counter (Counter Cooperation, Hialeah, FL). Diluted LN cells were injected through lateral tail vein to B6, Fas−/− or Prf−/− recipient mice at 4–10 × 106 cells/recipient in 400–500 μL Iscove's Modified Dulbecco's Medium. Recipients were preirradiated with 5, 6.5, or 7 G TBI using a 137Cesium γ source (J. L. Shepherd & Associates, Glandale, CA). Recipients were bled and euthanized 8–14 days after LN cell infusion to obtain tissues for histological and cytological analyses.Blood counts and flow cytometryBlood was collected from the retro-orbital sinus into ethylenediaminetetraacetic acid–added Eppendorf tubes. Complete blood counts (CBCs) were performed in a HemaVet 950 analyzer (Drew Scientific, Waterbury, CT). Plasma was separated by centrifugation at 8,000 g for 5 min and was stored at −30°C. After mouse euthanasia by CO2 inhalation, BM cells were extracted from bilateral tibiae and femurs, filtered through 90 μmol/L nylon mesh, and counted in a Vi-Cell counter. Peripheral blood leukocytes and BM cells were first incubated with Ack buffer twice for 10 min to lyse red blood cells (RBCs). Residual leukocytes were stained with various antibodies and analyzed on a LSR II or Canto II flow cytometer using the FACSDiva software (Becton Dickson, San Diego, CA). To measure cell apoptosis, cells were first stained with an antibody mixture along with Annexin V in specific high-calcium buffer using reagents from an Annexin V apoptosis detection kit from BD Biosciences (San Diego, CA) and were then added with 7AAD 10 minutes before data acquisition.Monoclonal antibodies for murine CD3 (clone 145-2C11), CD4 (clone GK 1.5), CD8 (clone 53-6.72), CD11a (clone 2D7), CD11b (clone M1/70), CD95 (Fas; clone Jo2), CD117 (c-Kit; clone 2B8), CD178.1 (FasL; clone MFL3), erythroid cells (clone Ter119), granulocytes (Gr1/Ly6-G; clone RB6-8C5), and stem cell antigen 1 (Sca-1; clone E13-161) were from BD Biosciences. The antimouse T-cell receptor β variable region antibody panel was also obtained from BD Biosciences. Antimouse CD45R (B220; clone RA3-6B2) was from Biolegend (San Diego, CA). Antibodies were conjugated to fluorescein isothiocyanate, phycoerythrin (PE), PE-cyanin 5 (PE-Cy5), PE-cyanin 7 (PE-Cy7), allophycocyanin (APC), or APC-cyanin 7 (APC-Cy7).Pathology and histologyMice treated with 6.5 G TBI + 4–10 × 106 FVB LN cells or with 6.5 G TBI only were euthanized at days 12–14. Lung, liver, kidney, intestine, spleen, and sternum were fixed in 10% neutral buffered formalin, sectioned at 5 μm thickness, and stained with H&E (VivoVitro Biotechnology, Rockville, MD). Slides were examined under a Zeiss Axioskop2 plus microscope and images were captured at 20× magnification using a Zeiss AxioCam HRC camera (Carl Zeiss MicroImaging, Jena, Germany).Luminex assays for plasma cytokinesA premixed 39-plex kit was obtained from R & D Systems (Minneapolis, MN). Plasma samples were filtered and loaded onto 96-well plates, then were incubated and washed according to the protocol from the manufacturer. A minimum of 50 beads per analyte was acquired. Median fluorescence intensities were collected on a Luminex-200 instrument using Bio-Plex Manager software version 6.2 (Bio-Rad Laboratories, Hercules, CA). Standard curves for each cytokine were generated using the premixed lyophilized standards provided in the kit. Cytokine concentrations in samples were determined from the standard curve using a 5-point regression to transform mean fluorescence intensities into concentrations. Each sample was run in duplicate, and the average of the duplicates was used as the measured concentration.StatisticsJMP statistical discovery software (SAS Institute, Cary, NC) was used to analyze CBC and BM cellular composition data through variance analysis with the compare all mean option for multiple comparisons [29SAS Institute IncJMP Statistics and Graphics Guide, Version 3. SAS Institute, Cary, NC1998Google Scholar]. Plasma cytokines were compared between TBI + FVB LN–treated and TBI-only animals using the Mann-Whitney test with Prism 6, as described earlier [30Feng X. Scheinberg P. Wu C.O. et al.Cytokine signature profiles in acquired aplastic anemia and myelodysplastic syndromes.Haematologica. 2011; 96: 602-606Crossref PubMed Scopus (107) Google Scholar]. For these analyses, statistical significance was declared at p < 0.05 and p < 0.01 respectively.ResultsInduction of bone marrow failure in B6 miceInfusion of 4–10 × 106 LN cells from FVB donors into normal B6 mice preirradiated with 6.5 G TBI (TBI + FVB LN) produced severe BM failure in recipients. We found that 43% (19/44) animals succumbed between 8 and 14 days following LN cell infusion, and surviving animals had severe pancytopenia and marrow hypoplasia with significant declines in neutrophils (p < 0.05) and platelets (p < 0.01; Fig. 1A), as well as in RBCs (p < 0.01) and total BM cells (p < 0.01; Fig. 1B), when compared with TBI-only animals or untreated controls. There were also declines in lymphocytes (p > 0.05), white blood cells (WBCs; p > 0.05), hemoglobin (p < 0.01), hematocrit (p < 0.01), and mean corpuscular volume (p < 0.01) in TBI + FVB LN–treated animals (data not shown). We were unable to induce marrow failure when we reduced the TBI dose to 5 G (TBI-L) or when we replaced FVB with BALB mice as LN cell donors: the TBI-L + BALB LN, TBI + BALB LN, and TBI-L + FVB LN treatment groups showed no cytopenia (Fig. 1C) and no change in total BM cells (Fig. 1D). We tested FVB LN cells at 4, 5, 8, and 10 × 106 cells per mouse, in combination with 6.5 G TBI. All cell doses were effective in producing marrow failure in B6 mice. Thus, we used 6.5 G TBI + 5 × 106 cells (TBI + FVB LN) as the standard regimen for induction of BM failure in B6 mice.To verify damage to HSCs and progenitors, we analyzed BM c-Kit+Sca-1+Lin− (KSL) cells. We observed that TBI + FVB LN treatment caused significant decline (p < 0.05) in the proportion of BM KSL cells (0.018 ± 0.006%) relative to those in untreated B6 controls (0.047 ± 0.006%; Fig. 1E). This change, along with a significant decrease in total BM cells, resulted in a sevenfold reduction in total BM KSL cells in BM failure mice (Fig. 1E).Clonal T cell expansionA characteristic feature of immune-mediated BM failure is T-cell-mediated destruction of BM hematopoietic cells. In this new model, we found greatly increased proportions of BM CD4 and CD8 T cells in TBI + FVB LN treated mice (Fig. 2A). On average, CD4 T cells increased eightfold (p < 0.01) and CD8 T cells increased sevenfold (p < 0.01) in the BM of TBI + FVB LN treated animals (Fig. 2B). Even considering the decline in total BM cells, there was a threefold (p < 0.05) increase in total CD4 cells and a twofold increase in total CD8 cells (p < 0.05) in TBI + FVB LN-treated animals relative to TBI-only controls (Fig. 2B). We further examined BM CD4 and CD8 T cell β variable region (Vβ) representation. Among the 15 Vβ groups, Vβ 7 (data not shown) and Vβ 17 were consistently upregulated in TBI + FVB LN treated animals (Fig. 2C). In CD4 T cells, Vβ 7 increased from 16.5 ± 4.7% to 23.4 ± 4.7% (p > 0.05), and Vβ 17a increased from 12 ± 1.9% to 21 ± 1.9% (p < 0.05) in TBI + FVB LN–treated animals. In CD8 T cells, the Vβ 7 proportion increased from 7.7 ± 2.3% to 28 ± 2.3% (p < 0.01), and the Vβ 17a proportion increased from 8.0 ± 1.8% to 22 ± 1.8% (p < 0.01), in TBI + FVB LN–infused animals relative to TBI-only controls (Fig. 2D). Overrepresentation of Vβ 7 and Vβ 17a CD4 and CD8 T cells indicated oligoclonal T-cell expansion in this immune-mediated BM failure model.Figure 2Oligoclonal T cell expansion. (A) Proportions and (B) total numbers of CD4 (p < 0.01 and p < 0.05) and CD8 (p < 0.01 and p < 0.05) T cells were significantly increased in the BM of mice that received TBI + FVB LN (n = 21) treatment compared with those that received TBI only (n = 12). (C) The expanded T cells in the BM of TBI + FVB LN–treated mice (n = 3) had distinctive Vβ 17 overrepresentation relative to TBI-only controls (n = 3). (D) Percentages of Vβ 7 (p < 0.01) and Vβ 17a (p < 0.01) CD8 T cells were significantly higher in the BM of TBI + FVB LN–treated animals than in TBI-only controls.View Large Image Figure ViewerDownload Hi-res image Download (PPT)T-cell activation and Fas-Fas ligand expressionIn addition to CD4 and CD8 T-cell expansion, there was also marked upregulation of T cell activation, as high proportions of CD4 and CD8 T cells expressing the T-cell activation marker CD11a (Fig. 3A). In the BM of TBI + FVB LN–treated animals, 96 ± 6.4% CD4 T cells and 98 ± 4.3% CD8 T cells were CD11a-positive, significantly higher (p < 0.01 and p < 0.05, respectively) than those in TBI only controls. In absolute terms, the total number of CD4+CD11a+ T cells was 6.5 times higher (p < 0.01), and the total number of CD8+CD11a+ T cells was 27 times higher (p < 0.01), in the BM of TBI + FVB LN–treated mice (Fig. 3A). T-cell CD11a expression was also upregulated in the peripheral blood of TBI + FVB LN–treated animals, although to a lesser degree than that seen in BM.Figure 3T cell activation and enhanced Fas/Fas ligand expression. (A) In addition to a significant T-cell expansion in the BM, B6 mice that received TBI + FVB LN treatment (n = 10) also had significantly higher proportions of CD11a+ cells, a marker of T-cell activation, in both CD4 (p < 0.01) and CD8 (p < 0.05) subsets, causing net gains of CD11a+CD4+ (p < 0.01) and CD11a+CD8+ (p < 0.01) T cells in the BM relative to TBI-only controls. (B) Residual BM cells from TBI + FVB LN treated mice (n = 3) showed elevated Fas expression (p < 0.01), and BM T cells, especially CD8 T cells, showed higher level of Fas ligand expression (p < 0.01) relative to BM cells from untreated control animals.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To define changes relevant to BM destruction, we examined the expression of Fas and FasL. Fas expression was upregulated in all BM cell fractions in TBI + FVB LN–treated animals, in which the number of Fas+ total BM cells was 60 ± 0.9%, 8.3 times higher (p < 0.01) than that in TBI only controls (7.2 ± 0.9%; Fig. 4B). In the fraction of expanded BM T cells from TBI + FVB LN–treated mice, 49 ± 2.5% CD8 T cells expressed FasL, twofold higher (p < 0.01) than the 25 ± 2.5% FasL-expressing BM CD8 T cells in TBI-only animals (Fig. 3B). FasL expression on expanded BM CD4 T cells, surprisingly, was not upregulated in BM failure animals (Fig. 3B). Overall, there were 3.8 times more (p < 0.01) Fas+ BM cells and 6.2 times more (p < 0.01) FasL+ CD8 T cells, in the BM of TBI + FVB LN–infused animals relative to TBI-only controls (Fig. 3B).Figure 4Elevation in hematopoietic cell apoptosis and BM destruction. BM cells from TBI + FVB LN–treated mice (n = 5) had significantly higher proportions of Annexin V+ (including both 7AADhigh and 7AADlow) apoptotic cells in KSL (p < 0.01), Lin− (p < 0.01), and whole BM (p < 0.01) cell fractions relative to TBI-only (n = 5) controls. (A) TBI + FVB LN–treated mice also had significantly higher proportions of Annexin V−7AADhigh dead cells in KSL (p < 0.01), Lin− (p < 0.01), and whole BM (p < 0.05) cells. Spleen, intestine, and sternum tissues from TBI-only (n = 5) and TBI + FVB LN–treated B6 mice (n = 6) were sectioned and hematoxylin & eosin stained for histological observations. (B) In comparison to TBI-only controls, TBI + FVB LN–treated mice had mild to moderate inflammation with lymphocyte infiltration in the spleen and intestines, along with severe BM damage showing empty marrow space.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Increased apoptosis and bone-marrow cell destructionUpregulation of Fas expression on BM cells and expansion of FasL+CD8 T cells suggested that the Fas/FasL pathway mediated BM destruction by increasing cell apoptosis. Indeed, BM cells from TBI + FVB LN–treated mice had significantly higher proportions of KSL (p < 0.01), Lin− (p < 0.01), and whole BM (p < 0.01) cells that entered apoptosis showing membrane binding to Annexin V (including both 7AADlow and 7AADhigh cells) relative to the same BM cell fractions from TBI-only animals (Fig. 4A). In addition, the proportion of Annexin V−7AADhigh dead cells was also significantly higher in KSL (p < 0.01), Lin−(p < 0.01), and whole BM (p < 0.05) cells from TBI + FVB LN–infused animals than in TBI-only controls (Fig. 4A).We observed that TBI + FVB LN cell infusion caused mild to moderate inflammation in the spleen and intestines, with disappearance of the germinal centers and mild infiltration of lymphocytes (Fig. 4B). However, the major pathology was the elimination of KSL cells, Lin− cells, and other cellular elements in the BM (Fig. 4B).Alterations in plasma cytokinesBM failure was associated with changes in blood plasma cytokine levels as measured in a 39-plex Luminex assay. Most notable were a twenty-sixfold increase in IFN-γ (p < 0.01) and a 34-fold increase in tumor necrosis factor α (TNFα; p < 0.01) in BM failure animals (Fig. 5A). Also significantly increased in the plasma of BM failure mice were the chemokine (C-C motif) ligands CCL3, at 112-fold increase (p < 0.01), CCL4 at sixfold (p < 0.01), CCL5 at twelve fold increase (p < 0.01), and CCL20 at elevenfold (p < 0.01), relative to TBI-only controls (Fig. 5B). Two chemokine (C-X-C motif) ligands, CXCL2 and CXCL5, increased by 154 times (p < 0.01) and 2.5 times (p < 0.05), respectively, in FVB LN–infused animals (Fig. 5C). Bone marrow failure mice also had significant elevations in hematopoietic growth factors: granulocyte colony–stimulating factor (G-CSF; 100 times, p < 0.01), granulocyte macrophage colony–stimulating factor (GM-CSF; 2.8 times, p < 0.05), macrophage colony–stimulating factor (M-CSF; 2.7 times, p < 0.01), and vascular endothelial growth factor (VEGF; 2.4 times, p < 0.01; Fig. 5D). There were also significant increases in 11 other plasma cytokine concentrations in BM failure mice relative to TBI-only controls (Table 1). Three cytokines, CCL21, insulin growth factor-1 (IGF-1), and interleukin (IL) 13, were significantly downregulated (Table 1), and 12 other cytokines, including IL-17a, showed no significant change during the development of BM failure (Table 2).Figure 5Upregulation in inflammatory cytokines, chemokines, and hematopoietic growth factors. Luminex measurements of blood plasma cytokine concentrations revealed significantly increased (A) inflammatory cytokines IFN-γ (p < 0.05) and TNF-α (p < 0.01); (B) chemokine (C-C motif) ligands CCL3 (p < 0.01), CCL4 (p < 0.01), CCL5 (p < 0.01), and CCL20 (p < 0.01); (C) chemokine (C-X-C motif) ligands CXCL2 (p < 0.01) and CXCL5 (p < 0.05); and (D) hematopoietic growth
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