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Background Immunosuppression therapy after lung transplantation fails to prevent bronchiolitis obliterans syndrome (BOS) in many patients, primarily a disease of the small airways. We have reported that BOS is associated with a lack of suppression of cytotoxic mediators, and proinflammatory cytokines, in peripheral blood T, NKT-like (particularly CD8+) and NK cells. We also showed a loss of glucocorticoid receptor (GCR) in proinflammatory lymphocytes after transplant. It is unknown whether these proinflammatory lymphocytes target the small and/or large airways in BOS. Methods Blood, bronchoalveolar lavage, large proximal, and small distal airway brushings were collected from patients with BOS (n = 10), stable lung transplant patients (n = 18), and healthy aged-matched controls (n = 10). Intracellular cytotoxic mediators (perforin/granzyme B), proinflammatory cytokines (IFNγ/TNFα), and expression of GCR were determined in lymphocytes subsets from cultured blood using flow cytometry. Results Increases in CD8 T cells, NKT-like cells, and NK cells were found in the small distal airways in BOS compared with stable patients and controls. An increase in perforin, granzyme B, IFNγ, TNFα, and a loss of GCR from these lymphocyte subsets was also found in BOS. GCR expression by CD8+ T cells from small airways correlated with FEV1 (R = 0.834, P = 0.039). Many of these changes significantly differed from those in the large airways. Conclusions BOS is associated with increased cytotoxic/proinflammatory CD8+ T, NKT-like, and NK cells in the small airways. Treatments that increase GCR in these lymphocyte subsets may improve graft survival.

BACKGROUND Immunosuppression therapy is ineffective at preventing chronic rejection of lung allografts (bronchiolitis obliterans syndrome [BOS]) and proinflammatory cytokines by steroid-resistant lymphocytes. The class III NAD-sirtuin 1 (SIRT1) is an important negative regulator of inflammation; however, SIRT1 activity following lung transplant has not been studied. We hypothesized that SIRT1 expression is decreased in proinflammatory lymphocytes following lung transplant and that treatment with SIRT1 activators (resveratrol, curcumin) and agents that prevent NAD depletion (theophylline) upregulate SIRT1 and reduce proinflammatory cytokine expression in these cells. METHODS Intracellular proinflammatory cytokines and SIRT1 were measured in blood T, natural killer T-like cell (NKT-like), and natural killer (NK) cells from patients with BOS (n = 10), stable lung transplant patients (n = 11), and healthy aged-matched controls (n = 10). Blood was cultured in the presence of ±25 µM resveratrol, ±1 µM curcumin, ±5 mg/L theophylline, ±1µM prednisolone and cytokines, and SIRT1 assessed using flow cytometry. RESULTS There was a loss of SIRT1 in T, NK-like, and NK cells in BOS patients compared with stable patients and controls (%CD8 SIRT1 T cells: 17 ± 10; 37 ± 10; 30 ± 10) (mean ± SEM BOS, stable, control, respectively) (P < 0.05 for all). Loss of SIRT1 was associated with increased T, NKT-like, and NK cells expressing interferon (IFN)γ and tumor necrosis factor (TNF)α. SIRT1 expression by T cells significantly associated with FEV1 (R = 0.655, P = 0.006) and with time posttransplant (R = -0.552, P = 0.041). All treatments upregulated SIRT1 and inhibited IFNγ and TNFα production by T, NK, and NKT-like cells additively. CONCLUSIONS BOS is associated with decreased SIRT1 in peripheral blood proinflammatory T, NK, and NKT-like lymphocytes following lung transplant. Treatment options that increase SIRT1 may improve graft survival.

Lymphocytic bronchiolitis (LB) has been shown to be an important factor for the subsequent development of obliterative bronchiolitis (OB). We have previously shown that OB, which limits long-term survival after lung transplantation, is associated with lack of suppression of peripheral blood T-cell granzyme B, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha. However, the role of these proinflammatory mediators in LB is unknown. We hypothesized that these proinflammatory mediators may also be increased during LB episodes despite standard immunosuppression regimens.T-cell intracellular cytokine profiles and granzyme B were studied in whole blood, bronchoalveolar lavage samples, and bronchial brushings from stable lung transplant patients with LB and from healthy controls, using multiparameter flow cytometry.There was a significant increase in peripheral blood T-cell granzyme B and CD8 T-cell IFN-gamma and TNF-alpha in patients with LB compared with control and stable groups and a decrease in CD25CD127CD3CD8 T regulatory cells in stable and LB transplant patients compared with controls. No changes were noted in the airways.LB is associated with inadequate suppression of peripheral blood T-cell granzyme B, IFN-gamma, and TNF-alpha. Drugs that effectively reduce these proinflammatory mediators may improve current protocols for treating LB and possibly reduce subsequent progression to OB in lung transplant patients.

BACKGROUND Allograft rejection remains a major cause of morbidity and mortality following lung transplantation and is associated with an increased expression of T-cell proinflammatory cytokines. We have recently shown that peripheral blood T-cell proinflammatory cytokine production was significantly reduced in stable lung transplant patients consistent with immunosuppression therapy. However, analysis of inflammatory cytokine profiles in bronchoalveolar lavage (BAL) T cells may be more relevant than peripheral blood T cells for assessing graft status. METHODS To investigate the immunomodulatory effects of currently used immunosuppressive regimens on BAL T-cell cytokine production, whole blood and BAL from stable lung transplant patients and control volunteers was stimulated in vitro and cytokine production by CD8+ and CD4+ T-cell subsets determined using multiparameter flow cytometry. RESULTS There was a significant decrease in T-cell proinflammatory cytokine production in BAL compared with blood from control subjects but not transplant patients. Anti-inflammatory cytokine IL-4 was increased in BAL compared with blood from both groups. There was a significant increase in IFNgamma, IL-2, IL-4, TGFbeta, and TNFalpha production by CD8 T cells and IFNgamma and TNFalpha production by CD4 T cells in BAL from transplant patients compared with controls. CONCLUSIONS We have shown decreased T-cell pro- and anti-inflammatory cytokine production in BAL compared with blood in control subjects but not in stable lung transplant patients. Current immunosuppression protocols have limited effect on T-cell proinflammatory cytokine production in BAL but do upregulate anti-inflammatory cytokines IL-4 and TGFbeta. Drugs that effectively reduce T-cell proinflammatory cytokine production in BAL may improve current protocols for reducing graft rejection in these patients.

BACKGROUND Alveolar macrophages are a major source of inflammatory cytokines and chemokines involved in the pathogenesis of lung transplant rejection and are derived from blood monocytes that migrate to the lung. Levels of monocyte cytokines and chemokines that may be relevant in transplant rejection have not previously been determined in transplant recipients. We hypothesized that production of these inflammatory mediators by blood monocytes may be up-regulated despite the use of potent immunosuppression therapy. METHOD To investigate this, whole blood from 9 stable lung transplant recipients and 12 control volunteers was stimulated with lipopolysaccharide in vitro, and intracellular chemokine and cytokine production were determined with multiparameter flow cytometry. RESULTS Monocyte production of chemokines interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, and MCP-3, and anti-inflammatory cytokine IL-10 were significantly increased in the lung transplant group, but IL-6, tumor necrosis factor-alpha, IL-1alpha, IL-12, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and transforming growth factor-beta levels were unchanged. CONCLUSIONS Because MCP-3 is a major chemoattractant for leukocytes to sites of antigenic challenge and is a natural ligand for MCP-1 receptor, this novel finding has important implications for the pathogenesis of lung transplant rejection. We now provide evidence that current immunosuppression protocols have a limited effect on monocyte inflammatory cytokine production and do not adequately suppress monocyte IL-8, MCP-1, and MCP-3 chemokine production. Drugs that modulate the action of these chemokines may improve current protocols for reducing graft rejection. Intracellular chemokine and cytokine analysis with flow cytometry may be a more accurate indicator of immunosuppression than drug levels in these patients.