The following abstracts were submitted in a timeframe that did not permit publication in Vol. 53 No. 2 of the Journal of Investigative Medicine. They were presented at the 2005 Combined Annual Meeting in Chicago, Illinois, April 15, 2005.
72 REGULATION OF MACROPHAGE SURVIVAL. D.M. FLAHERTY, S.L. HINDE, M.M. MONICK, G.W. HUNNINGHAKE, DEPARTMENT OF INTERNAL MEDICINE, UNIVERSITY OF IOWA, ROY J. AND LUCILLE A. CARVER COLLEGE OF MEDICINE, IOWA CITY, IA.
Human alveolar macrophages play a critical role in host defense and in the development of inflammation and fibrosis in the lung. Unlike their precursor cells, blood monocytes, alveolar macrophages are long-lived and tend to be resistant to apoptotic stimuli. We hypothesized that one result of differentiation of blood monocytes into alveolar macrophages is an alteration in the constitutive expression of survival pathway activity. Using Western analysis for phoshorylation specific activity sites, we found high levels of PI3-kinase/Akt and ERK activity in human alveolar macrophages. In contrast, monocyte levels of Akt and ERK activity at baseline were very low. In evaluating a possible reason for the high Akt, we found that baseline levels of PTEN, a tumor suppressor protein with lipid phosphatase activity against phosphatidylinositol 3,4,5 trisphosphate (PI3,4,5P3), the major bioactive product of PI3-kinase, were very low in alveolar macrophages. Opposite to the Akt activity findings, PTEN levels in blood monocytes were very high. These findings were duplicated using in vitro differentiation of monocytes to macrophages. To determine the survival consequences of the high Akt and ERK activity in alveolar macrophages, we cultured freshly isolated cells with the PI3-kinase inhibitor, LY294002, the ERK inhibitor, U0126, or both, in combination. Inhibition of either Akt or ERK resulted in shortened survival of the alveolar macrophages, but combining the two inhibitors significantly increased cell death. In conclusion, we demonstrate that prolonged survival of alveolar macrophages requires high levels of Akt and ERK activity which are modulated during the differentiation process.
73 ABNORMAL PHENOTYPE AND JUNCTION OF MYELOID DENDRITIC CELLS IN SYSTEMIC LUPUS ERYTHEMATOSUS. M.J. KAPLAN, UNIVERSITY OF MICHIGAN, ANN ARBOR, MI.
Systemic lupus erythematosus (SLE) is characterized by a systemic autoimmune response with profound and diverse T and B cell changes. It has been proposed that these abnormalities could be explained by alterations in dendritic cell (DC) function, since these are key regulators of the immune system. Methods: 86 SLE patients, 16 patients with rheumatoid arthritis and 30 healthy controls were studied. Myeloid DCs were derived from PBMCs by IL-4/GM-CSF stimulation for 7 days. To induce maturation, LPS and/or TNF-α were added for additional 48 h. Cells were studied at 10 days, or co-cultured with T cells for 18 hr. CD1a, CD83, CD86 and HLA-DR expression were evaluated by flow cytometry and CD83, CD80, CD11c, CD1a, CD86, HLA-DR, TNF-α and IL-12 mRNA were detected by multiplex RT-PCR. Secreted cytokines were measured by multiplex ELISA. Apoptosis was measured by Annexin V FITC expression. Allogeneic T cell proliferation was measured by CFSE. Results: At 7 days, CD1a mRNA and protein expression was significantly higher in lupus DCs than in healthy controls. Interestingly, cell surface and mRNA expression of the maturation marker CD83 was lower in DCs from SLE patients than in controls, suggesting an intrinsic maturation defect (20% + 2.99 vs 52% + 8.3, p < .05 for protein expression. However, the expression of other maturation markers was significantly higher in the SLE group, suggesting a selective decrease in CD83. While SLE DCs upregulate maturation markers in response to LPS or TNF-α, their response was blunted when compared to healthy controls. These changes in differentiation and maturation significantly correlated with disease activity and the presence of lupus nephritis (p < .01). In addition, SLE DCs display a specific cytokine profile, with an increase in IL-8 production, and promote differential allogeneic T cell stimulation. Conclusions: Taken together, our data suggest that monocyte-derived DC differentiation and maturation are altered in SLE. We hypothesize that these events could contribute to initiate and maintain the autoimmune response in lupus. The functional correlations and consequences of these phenotypic abnormalities remain to be determined. Funded by NIAMS.
74 ROLE OF ANTIGEN PRESENTING CELLS IN GRAFT VERSUS LEUKEMIA EFFECT AFTER ALLOGENEIC BONE MARROW TRANSPLANTATION. P.R. REDDY, UNIVERSITY OF MICHIGAN, ANN ARBOR, MI.
The graft-versus-leukemia/tumor (GVL) effect after allogeneic bone marrow transplantation (BMT) is tightly linked to acute graft-versus-host disease (GVHD). Recent data demonstrate that donor T cells along with host and donor antigen presenting cells (APCs) and a network of pro-inflammatory cytokines are necessary and sufficient for induction of GVHD. But the roles of APCs and allo-antigen expression in GVL remain ill-defined. We utilized well defined murine models of bone marrow chimeras of GVHD and GVL to determine the role of APCs in GVL. Bone marrow chimeric mice were generated with and without class I on their APCs but with class I on target tissues [B6′B6] and B6′ [β2M-/-′B6], respectively. After complete hematopoietic turnover these chimeras were re-irradiated and injected with host specific tumor cells (MBL, H-2b, murine lymphoma cells) along with CD8+ T cells and T cell depleted bone marrow (TCD BM) from either allogeneic C3H.SW (that differ from the hosts across multiple minor histocompatibility antigens) or syngeneic (B6) donors. All of the syngeneic mice B6′[B6′B6] and B6′ [β2M-/-′B6] died from tumor without GVHD. The allogeneic C3H.SW' [B6′B6] recipients demonstrated absence of tumor but died from GVHD. In contrast the allogeneic C3H.SW'[β2M-/-′B6] recipients died from tumor without signs of GVHD. We next determined the role pro-inflammatory cytokines, expression of allo-antigens on APCs, target and tumor tissues in GVL. To this end [B6′B6], [bm1′B6], [B6′bm1] and [bm1′bm1] chimeras were generated where B6 mice differ from the B6.C-H-2bm1 (bm1) mice across a single class I antigen. These animals were re-irradiated and injected with MBL (H-2b) tumor cells along with TCD BM and CD8+ cells from either B6 or bm1 donors. The results from these experiments demonstrated that (a) allogeneic APCs alone are insufficient to mediate GVL; (b) tumor tissue allo-antigen expression is required for GVL; (c) host APCs mediate a more robust GVL response that donor type APCs. Together our results provide a complete picture of the roles of APCs and allo-antigen expression in mediating GVL.