Neutralizing IL-17 prevents obliterative bronchiolitis in murine orthotopic lung transplantation.

Obliterative bronchiolitis (OB) is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining OB immunopathogenesis. In the current study, lungs from C57BL/10 H-2(b) mice that are MHC compatible, but minor histocompatability antigen incompatible, were transplanted into C57BL/6 mice. Histological features and cytokine profiles of OB were assessed. Moderate rejection (grade A3) developed by day 14, with evidence of OB at that time point. At 21 days, OB was present in 55% of grafts and moderate to severe rejection (grade A3-A4) was present in all mice. At 28 days, OB was present in 44% of mice and severe rejection (grade A4) was present in all. IL-17A, but not IL-17F, splenic mRNA transcripts and serum protein levels were increased only in mice that developed OB, whereas IL-10 transcripts and protein were increased only in non-OB mice. Neutralizing IL-17 prevented OB, down regulated acute rejection, and upregulated systemic IL-10. Collectively, these data show that transplantation of minor histoincompatible lungs from C57BL/10 mice into C57BL/6 mice results in a highly reproducible preclinical model of OB. In addition, these data indicate that neutralizing IL-17A or augmenting IL-10 could be therapeutic interventions to prevent OB.

Silencing S1P1 receptors regulates collagen-V reactive lymphocyte-mediated immunobiology in the transplanted lung.

Type V collagen (col[V])-reactive lymphocytes contribute to lung transplant rejection, but the mechanisms for emigration into the graft are unknown. Sphingosine-1-phosphate-1 receptors (S1P(1R)) are believed to be required for lymphocyte emigration in other studies, but their role in col(V)-reactive lymphocyte rejection responses is not known. Utilizing small interfering RNA (siRNA) to reduce S1P(1R) expression on col(V)-reactive lymphocytes, we examined the role of S1P(1R) in the rejection response. Quantitative polymerase chain reaction (PCR) revealed strong expression of S1P(1R) messenger RNA (mRNA)on col(V)-reactive lymphocytes isolated from immunized rats. S1P(1R)-specific siRNA (S1P(1R) siRNA) reduced expression of S1P(1R) mRNA and protein, whereas scramble siRNA (SC siRNA) had no effect. Adoptive transfer of lymphocytes treated with S1P(1R) siRNA to rat Wistar Kyoto (WKY) lung isograft recipients resulted in retention of cells within the liver with fewer cells in mediastinal lymph nodes when compared to cells exposed to SC siRNA. S1P(1R)-deficient cells proliferated in response to alloantigens, but not in response to col(V), and produced less interferon (IFN)-gamma in response to col(V) compared to controls. Downregulating S1P(1R) did not affect production of interleukin (IL)-10and tumor necrosis factor (TNF)-alpha, or expression of adhesion molecules critical for migration, but prevented rejection pathology and lowered local levels of IFN-gamma post adoptive transfer. These data demonstrate novel roles of S1P(1R,) which include regulating emigration and modulating lymphocyte activation.

Anti-type V collagen lymphocytes that express IL-17 and IL-23 induce rejection pathology in fresh and well-healed lung transplants.

Immunity to collagen V [col(V)] contributes to lung 'rejection.' We hypothesized that ischemia reperfusion injury (IRI) associated with lung transplantation unmasks antigenic col(V) such that fresh and well-healed lung grafts have differential susceptibility to anti-col(V)-mediated injury; and expression of the autoimmune cytokines, IL-17 and IL-23, are associated with this process. Adoptive transfer of col(V)-reactive lymphocytes to WKY rats induced grade 2 rejection in fresh isografts, but induced worse pathology (grade 3) when transferred to isograft recipients 30 days post-transplantation. Immunhistochemistry detected col(V) in fresh and well-healed isografts but not native lungs. Hen egg lysozyme-reactive lymphocytes (HEL, control) did not induce lung disease in any group. Col(V), but not HEL, immunization induced transcripts for IL-17 and IL-23 (p19) in the cells utilized for adoptive transfer. Transcripts for IL-17 were upregulated in fresh, but not well-healed isografts after transfer of col(V)-reactive cells. These data show that IRI predisposes to anti-col(V)-mediated pathology; col(V)-reactive lymphocytes express IL-17 and IL-23; and anti-col(V)-mediated lung disease is associated with local expression of IL-17. Finally, because of similar histologic patterns, the pathology of clinical rejection may reflect the activity of autoimmunity to col(V) and/or alloimmunity.

Oral tolerance induction by type V collagen downregulates lung allograft rejection.

Immunization with specific proteins or peptides has been used to induce immunologic tolerance to allografts other than the lung. Recently, we have reported that the immune response to lung alloantigen also involves an immune response to type V collagen [col(V)]. The purpose of the current study was to determine if oral administration of col(V) to lung allograft recipients before transplantation downregulates acute rejection episodes. The data show that, compared with controls, col(V)-fed recipients had fewer polymorphonuclear cells and lymphocytes in allograft bronchoalveolar lavage fluid, and reduced rejection pathology. Data showing that col(V)- fed allograft recipients had diminished delayed-type hypersensitivity (DTH) responses to donor alloantigens suggest that feeding col(V) prevented allograft rejection by inducing tolerance to donor antigens. Systemic production of transforming growth factor (TGF)-beta, interleukin (IL)-4, or IL-10 has been reported to be a mechanism for oral tolerance-induced suppression of immune responses. Feeding col(V) induced upregulated production of TGF-beta, but not IL-4 or IL-10 in serum. Neutralizing TGF-beta recovered the DTH response to donor antigen in tolerant allograft recipients. Collectively, these data show that oral administration of col(V) is a novel approach to induce immunologic tolerance to lung allografts, and that TGF-beta contributed to suppression of the rejection response.

Monocyte chemoattractant protein-1 and RANTES are chemotactic for graft infiltrating lymphocytes during acute lung allograft rejection.

Graft infiltrating lymphocytes (GILs) are crucial to rejection of lung allografts. However, chemotactic activities, chemokines responsible for GIL recruitment, and cells involved in chemokine production during lung allograft rejection have not been evaluated. This study determined whether chemotactic activity for GILs is upregulated, and whether the chemokines monocyte chemoattractant protein (MCP)-1 and regulated on activation, normal T cells expressed and secreted (RANTES) have roles in GIL chemotaxis during lung allograft rejection. F344 (RT1(lv1)) rat lung allografts were transplanted into WKY (RT1(l)) recipients. Chemotactic activity for GILs and quantities of MCP-1 and RANTES were determined in allograft bronchoalveolar lavage fluid 1 wk after transplantation. Data showed that during rejection, chemotactic activity for GILs is upregulated, MCP-1 and RANTES are produced locally, and both MCP-1 and RANTES are operative in GIL recruitment. Immunohistochemistry showed that alveolar macrophages (AMs) were the major source of MCP-1 and that other lung cells, including AMs, were the source of RANTES. Further, depletion of AMs in the donor lung before transplantation downregulated chemotaxis for GILs and production of MCP-1 during rejection episodes. These data show that chemotaxis for GILs is upregulated locally during lung allograft rejection, and that MCP-1 and RANTES contribute to GIL recruitment during the rejection response.

Instillation of allogeneic lung antigen-presenting cells deficient in expression of major histocompatibility complex class I or II antigens have differential effects on local cellular and humoral immunity and on pathology in recipient murine lungs.

Recognition of allogeneic major histocompatibility complex (MHC) molecules expressed on donor lung antigen-presenting cells (APCs) by host T lymphocytes is believed to stimulate lung allograft rejection. However, the specific roles of donor MHC molecules in the rejection response is unknown. We report a murine model in which instilling allogeneic lung APCs into recipient lungs induces pathology analogous to acute rejection, and the production of interferon (IFN)-gamma, immunoglobulin (Ig) G2a, and alloantibodies in recipient lungs. Using allogeneic lung APCs (C57BL/6, I-a(b), H-2(b)) deficient in MHC class I, II, or both for instillation into lungs of BALB/c mice (I-a(d), H-2(d)), the purpose of the current study was to determine the specific roles of donor MHC molecules in stimulating local alloimmune responses. The data show that MHC class I or II on donor APCs induced IFN-gamma and IgG2a synthesis locally, though less than that induced by wild-type cells. Both MHC class I and II were required to induce alloantibody production. Instillation of wild-type or class I- or class II-deficient APCs induced comparable pathologic lesions in recipient lungs, and more severe than that induced by MHC-deficient cells. These data show that donor MHC class I and II molecules have differential effects in the stimulation of local alloimmune responses.

Type V collagen modulates alloantigen-induced pathology and immunology in the lung.

Perivascular and peribronchiolar tissues are targets of the immune response during lung allograft rejection. Collagen type V (col[V]) is located within these tissues. Col(V) may be major histocompatibility complex (MHC)-like, and MHC-derived peptides have been used to induce immunologic tolerance and prevent rejection in allografts other than the lung. The current study tests the hypothesis that col(V) could be used to downregulate immune responses to lung alloantigen in vivo. We developed a murine model in which instillations of allogeneic bronchoalveolar lavage (BAL) cells (C57BL/6, I-a(b), H-2(b)) into lungs of BALB/c mice (I-a(d), H-2(d)) induce histology similar to grades 1 and 2 acute lung allograft rejection, apoptosis of airway epithelium and vascular endothelium, and upregulate tumor necrosis factor (TNF)-alpha production locally. The current study reports that instillations of col(V) into lungs before allogeneic BAL cells prevent development of rejection pathology and apoptosis, downregulate alloantigen-induced T-lymphocyte proliferation, and abrogate local TNF-alpha production. In addition, instillation of col(V)-pulsed autologous BAL cells into lungs of mice primed with allogeneic BAL cells perpetuates rejection pathology. Collectively, these data show that col(V) is a novel antigen involved in the rejection process, and suggest that col(V) could be used to modulate the rejection response to lung allografts.

Allogeneic bronchoalveolar lavage cells induce the histology and immunology of lung allograft rejection in recipient murine lungs: role of intercellular adhesion molecule-1 on donor cells.

BACKGROUND: Intercellular adhesion molecule (ICAM)-1 expressed on accessory cells has a key role in antigen presentation. The histology and immunology of lung allograft rejection is postulated to result from donor lung accessory cells presenting alloantigens to recipient lymphocytes, and, therefore, ICAM-1 may have a crucial role in the rejection process. We have previously reported that the instillation of allogeneic (C57BL/6, I-a(b)) bronchoalveolar lavage (BAL) cells (96% macrophages, 2% dendritic cells) into the lungs of recipient BALB/c mice (I-a(d)) induced the histology and immunology of acute lung allograft rejection. Using this model, the purpose of the current study was to determine the role of ICAM-1 on donor lung cells in lung allograft rejection. METHODS: BALB/c mice received allogeneic BAL cells from wild-type or ICAM-1 mutant (lacking ICAM-1 expression) C57BL/6 mice by nasal insufflation weekly for 4 weeks. Recipient mice underwent BAL and serum collection for the determination of T helper 1/T helper 2 cytokines and IgG subtypes. Lung histology was graded using standard criteria for allograft rejection. RESULTS: Although wild-type cells induced a lymphocytic vasculitis and bronchitis, ICAM-1 mutant allogeneic BAL cells only induced a lymphocytic vasculitis in recipient lungs. Both wild-type and ICAM-1 mutant cells induced up-regulated local interferon-gamma and IgG2a production, and deposition of IgG2a in recipient lungs. CONCLUSIONS: These data show that ICAM-1 on donor lung accessory cells mediates differential effects on the histology and immunology of acute lung allograft rejection.

Instillation of allogeneic lung macrophages and dendritic cells cause differential effects on local IFN-gamma production, lymphocytic bronchitis, and vasculitis in recipient murine lungs.

Lung allograft rejection is believed to be initiated by donor lung accessory cells, namely macrophages and dendritic cells, interacting with recipient lymphocytes leading to up-regulated Th1 type (IFN-gamma) cellular immunity culminating in graft destruction. The purpose of this study was to determine the individual role of donor lung macrophages and dendritic cells in the rejection response. Utilizing a murine model that reproduces the immunology and histology of acute rejection, C57BL/6 mouse (I-a(b), H-2(b)) lung dendritic cells (DC-enriched lung cells), purified alveolar macrophages (I-a-negative macrophages), or various ratios of I-a-negative macrophages/DC were instilled into BALB/c mouse (I-a(d), H-2(d)) lungs followed by an assessment of local IFN-gamma production and grading of rejection pathology. The data show that DC, and not I-a-negative macrophages, induced IFN-gamma production in recipient lungs. However, the local production of IFN-gamma was not always associated with histological changes characteristic of rejection pathology. In contrast to either cell type alone, instillation of C57BL/6 I-a-negative macrophages and DC, together, were required to induce rejection pathology in BALB/c lungs. In addition, the rejection response was dependent on interactions between donor I-a-negative macrophages and DC.

Role of passenger leukocytes in allograft rejection: effect of depletion of donor alveolar macrophages on the local production of TNF-alpha, T helper 1/T helper 2 cytokines, IgG subclasses, and pathology in a rat model of lung transplantation.

Acute lung allograft rejection is believed to be initiated by passenger leukocytes, such as alveolar macrophages (AM), in the donor organ, which release TNF-alpha, and present alloantigens to host lymphocytes, to up-regulated Th1 cellular and humoral immunity. However, the role of donor AM in local TNF-alpha synthesis, and their ability to induce local Th1 cellular and humoral immunity have not been evaluated. By depleting Brown Norway (BN, RT1n) rat lung allografts of AM before transplantation into Lewis rat (LEW, RT1(1)) recipients, the current study determined the role of donor AM in including the production of TNF-alpha, IFN-gamma (Th1 cytokine), IL-4 (Th2 cytokine), IgG subtypes, and rejection pathology in the allograft. The data show that compared with untreated BN allografts, pretransplant depletion of donor lung AM resulted in significantly less TNF-alpha, and IFN-gamma production in allograft bronchoalveolar lavage fluid with variable effects on local IL-4 production. Depletion of AM in the donor lung before transplantation affected the local production of several IgG subclasses. However, pretransplant depletion of donor AM had no effect on the development of the pathology of severe acute rejection. These data show that donor AM have a central role in the local synthesis of TNF-alpha and induce the production of IFN-gamma and IgG subtypes, locally, during acute lung allograft rejection. However, depletion of AM before transplantation does not prevent the development of severe acute rejection in BN rat lungs, transplanted into LEW recipients.

Impaired IgG production in the lungs of HIV-infected individuals.

Human immunodeficiency virus (HIV)-infected individuals are at risk for pulmonary infections with encapsulated bacterial pathogens. This could reflect impaired production of opsonizing antibodies in the lower respiratory tract. We examined antibody production in the alveolar space by measuring immunoglobulin concentrations in bronchoalveolar lavage (BAL) of HIV-infected patients and normal volunteers and by assessing the ability of alveolar macrophages (AM) to induce immunoglobulin production in normal peripheral blood mononuclear cells (PBMC). BAL from HIV-infected patients contained significantly less IgG than normal BAL. IgA and IgM concentrations were similar in both groups. Normal AM supported IgG and IgA production in PBMC. While HIV AM could induce IgA production in PBMC, in no instance did they induce IgG secretion. HIV AM produced significantly more transforming growth factor-beta (TGF-beta), a factor known to suppress IgG production, than normal AM. Finally, TGF-beta antibodies blocked the inhibitory effect of HIV AM on normal IgG secretion without affecting IgA secretion. These findings demonstrate impaired production of opsonizing IgG in the alveolar space of HIV-infected subjects and implicate excess TGF-beta production by AM as the cause of this impairment.

Effect of human lung allograft alveolar macrophages on IgG production: immunoregulatory role of interleukin-10, transforming growth factor-beta, and interleukin-6.

Alveolar macrophages (AM) are crucial to initiating and maintaining local immune responses. The increased susceptibility to pulmonary infections in lung allograft recipients may be due to impaired AM function resulting in diminished cellular and humoral immunity. We have previously reported that control AM were potent stimulators of IgG production from allogeneic peripheral blood mononuclear cells (PBM) in a manner that was dependent on gamma-interferon (gamma IFN). The ability of allograft AM to induce IgG production is unknown. The purpose of the current study was to compare the ability of allograft and control AM to induce IgG production from allogeneic PBM. In contrast to control AM which induced a dose-dependent stimulation of IgG production from allogeneic PBM, allograft AM were highly suppressive of IgG production. The inhibition was not due to a lack of allograft AM stimulation of gamma IFN production from responding lymphocytes. Supernatants from allograft AM were highly suppressive of control AM-induced IgG production. Allograft AM produced greater quantities of interleukin (IL-10) than control AM while transforming growth factor-beta (TGF-beta) production from these cells was comparable. Blocking antibodies to IL-10 and TGF-beta reversed the inhibition of IgG production to 63% and 60% of control, respectively. In addition, the production of interleukin 6 (IL-6), a macrophage-derived cytokine crucial to the stimulation of IgG synthesis, was deficient in the allograft AM. Addition of IL-6 to allograft AM and allogeneic PBM co-cultures restored IgG synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Allogeneic bronchoalveolar lavage cells induce the histology of acute lung allograft rejection, and deposition of IgG2a in recipient murine lungs.

The immunologic and histologic changes associated with lung allograft rejection are believed to result from the presentation of donor lung alloantigens to recipient lymphocytes resulting in up-regulated Th1 lymphocyte activity. The ability of allogeneic lung immune cells to induce the pathologic and immunologic changes associated with acute lung allograft rejection are unknown. The current study determined whether allogeneic (C57BL/6, I-a(b)) bronchoalveolar lavage (BAL) cells (> or = 97% macrophages), when instilled into the lungs of recipient BALB/c mice (I-a(d)), induced the histology and immunology associated with acute lung allograft rejection. BALB/c mice received BAL cells from either C57BL/6 mice (allogeneic instillate) or BALB/c mice (autologous instillate) or PBS (control) by nasal insufflation weekly for 4 wk. Allogeneic BAL cells resulted in a lymphocytic bronchitis and vasculitis analogous to grade 1 to 2 lung allograft rejection. The mice given allogeneic instillates had a greater percentage of lymphocytes in the BAL fluid than those given autologous instillates. After instillation of allogeneic BAL cells, the Th1 cytokines, IL-2 and IFN-gamma (IFN-gamma), were produced locally in greater quantities and more frequently than Th2 cytokine IL-10. IL-4, another Th2 cytokine, was not detected. The local production of IgG1 and IgG2a, which are dependent on IL-4 and IFN-gamma, respectively, were increased. However, only IgG2a was deposited in the perivascular and peribronchiolar tissues. These data show that installation of allogeneic BAL cells into the airways of recipient mice induced up-regulated Th1 lymphocyte activity and caused the histologic changes associated with lung allograft rejection.

Increased bronchoalveolar IgG2/IgG1 ratio is a marker for human lung allograft rejection.

BACKGROUND: Lung allograft rejection (AR) is thought to involve T-helper-1 (Th-1) lymphocytes mediating both cellular immunity and alloantibody production. Th-1 lymphocytes produce gamma interferon (gamma IFN) and induce IgG2 production, suggesting that increased IgG2 production might occur during AR. The purpose of this study was to determine if locally altered bronchoalveolar IgG2/IgG1 ratios might correlate with AR. METHODS: Eighteen recipients of lung allografts underwent a total of 25 bronchoscopies for surveillance or at times of suspected infection or AR. Bronchoalveolar lavage (BAL), serum collection, and transbronchial biopsy (TB) were performed on all patients. gamma IFN, IgG1, IgG2 levels, and the ratio of IgG2/IgG1 were determined in serum and BAL and matched with TB histology. Five nonsmoking normal volunteers undergoing bronchoscopy, BAL, and serum collection served as controls. RESULTS: IgG2 was upregulated in allograft BAL during AR as determined by the ratio IgG2/IgG1 (2.91 +/- 0.79 SEM vs 0.62 +/- SEM, p < 0.019, IgG2/IgG1, AR BAL vs non-AR BAL, respectively). An IgG2/IgG1 > or = 1 in allograft BAL (95% confidence intervals 1.26 to 4.56) was 80% specific and 91% sensitive for the diagnosis of AR with a positive predictive value of 92%. A BAL IgG2/IgG1 < 1 (95% confidence interval 0.27 to 0.97) had a negative predictive value of 77%. After therapy in two patients the elevated IgG2/IgG1 ratio reversed to normal (ie, < 1) with histologic resolution of AR. CONCLUSIONS: Human lung AR is associated with a locally increased IgG2/IgG1 ratio suggesting locally upregulated Th-1 lymphocyte activity during lung AR.