Th17 Responses to Collagen Type V, kα1-Tubulin, and Vimentin Are Present Early in Human Development and Persist Throughout Life.

T helper 17 (Th17)-dependent autoimmune responses can develop after heart or lung transplantation and are associated with fibro-obliterative forms of chronic rejection; however, the specific self-antigens involved are typically different from those associated with autoimmune disease. To investigate the basis of these responses, we investigated whether removal of regulatory T cells or blockade of function reveals a similar autoantigen bias. We found that Th17 cells specific for collagen type V (Col V), kα1-tubulin, and vimentin were present in healthy adult peripheral blood mononuclear cells, cord blood, and fetal thymus. Using synthetic peptides and recombinant fragments of the Col V triple helical region (α1[V]), we compared Th17 cells from healthy donors with Th17 cells from Col V-reactive heart and lung patients. Although the latter responded well to α1(V) fragments and peptides in an HLA-DR-restricted fashion, Th17 cells from healthy persons responded in an HLA-DR-restricted fashion to fragments but not to peptides. Col V, kα1-tubulin, and vimentin are preferred targets of a highly conserved, hitherto unknown, preexisting Th17 response that is MHC class II restricted. These data suggest that autoimmunity after heart and lung transplantation may result from dysregulation of an intrinsic mechanism controlling airway and vascular homeostasis.

Proteomic Characterization Reveals That MMP-3 Correlates With Bronchiolitis Obliterans Syndrome Following Allogeneic Hematopoietic Cell and Lung Transplantation.

Improved diagnostic methods are needed for bronchiolitis obliterans syndrome (BOS), a serious complication after allogeneic hematopoietic cell transplantation (HCT) and lung transplantation. For protein candidate discovery, we compared plasma pools from HCT transplantation recipients with BOS at onset (n = 12), pulmonary infection (n = 16), chronic graft-versus-host disease without pulmonary involvement (n = 15) and no chronic complications after HCT (n = 15). Pools were labeled with different tags (isobaric tags for relative and absolute quantification), and two software tools identified differentially expressed proteins (≥1.5-fold change). Candidate proteins were further selected using a six-step computational biology approach. The diagnostic value of the lead candidate, matrix metalloproteinase 3 (MMP3), was evaluated by enzyme-linked immunosorbent assay in plasma of a verification cohort (n = 112) with and without BOS following HCT (n = 76) or lung transplantation (n = 36). MMP3 plasma concentrations differed significantly between patients with and without BOS (area under the receiver operating characteristic curve 0.77). Consequently, MMP3 represents a potential noninvasive blood test for diagnosis of BOS.

Objective Estimates Improve Risk Stratification for Primary Graft Dysfunction after Lung Transplantation.

Primary graft dysfunction (PGD) is a major cause of early mortality after lung transplant. We aimed to define objective estimates of PGD risk based on readily available clinical variables, using a prospective study of 11 centers in the Lung Transplant Outcomes Group (LTOG). Derivation included 1255 subjects from 2002 to 2010; with separate validation in 382 subjects accrued from 2011 to 2012. We used logistic regression to identify predictors of grade 3 PGD at 48/72 h, and decision curve methods to assess impact on clinical decisions. 211/1255 subjects in the derivation and 56/382 subjects in the validation developed PGD. We developed three prediction models, where low-risk recipients had a normal BMI (18.5-25 kg/m(2) ), chronic obstructive pulmonary disease/cystic fibrosis, and absent or mild pulmonary hypertension (mPAP<40 mmHg). All others were considered higher-risk. Low-risk recipients had a predicted PGD risk of 4-7%, and high-risk a predicted PGD risk of 15-18%. Adding a donor-smoking lung to a higher-risk recipient significantly increased PGD risk, although risk did not change in low-risk recipients. Validation demonstrated that probability estimates were generally accurate and that models worked best at baseline PGD incidences between 5% and 25%. We conclude that valid estimates of PGD risk can be produced using readily available clinical variables.

Differential requirement for P2X7R function in IL-17 dependent vs. IL-17 independent cellular immune responses.

IL17-dependent autoimmunity to collagen type V (Col V) has been associated with lung transplant obliterative bronchiolitis. Unlike the T helper 1 (Th1)-dependent immune responses to Tetanus Toxoid (TT), the Th17 response to Col V in lung transplant patients and its Th1/17 variant observed in coronary artery disease patients requires IL-1ß, tumor necrosis factor α and CD14(+) cells. Given the involvement of the P2X7 receptor (P2X7R) in monocyte IL-1ß responses, we investigated its role in Th17-, Th1/17- and Th1-mediated proinflammatory responses. Transfer of antigen-pulsed peripheral blood mononucleated cells (PBMCs) from Col V-reactive patients into SCID mouse footpads along with P2X7R antagonists revealed a selective inhibition of Col V-, but not TT-specific swelling responses. P2X7R inhibitors blocked IL-1ß induction from monocytes, including both Col V-α1 peptide-induced (T-dependent), as well as native Col V-induced (T-independent) responses. Significantly higher P2X7R expression was found on CXCR3(neg) CCR4(+)/6(+) CD4(+) [Th17] versus CXCR3(+)CCR4/6(neg) CD4(+) [Th1] subsets in PBMCs, suggesting that the paradigm of selective dependence on P2X7R might extend beyond Col V autoimmunity. Indeed, P2X7R inhibitors suppressed not only anti-Col V, but also Th1/17-mediated alloimmunity, in a heart transplant patient without affecting anti-viral Epstein-Barr virus responses. These results suggest that agents targeting the P2X7R might effectively treat Th17-related transplant pathologies, while maintaining Th1-immunity to infection.

Preoperative plasma club (clara) cell secretory protein levels are associated with primary graft dysfunction after lung transplantation.

Inherent recipient factors, including pretransplant diagnosis, obesity and elevated pulmonary pressures, are established primary graft dysfunction (PGD) risks. We evaluated the relationship between preoperative lung injury biomarkers and PGD to gain further mechanistic insight in recipients. We performed a prospective cohort study of recipients in the Lung Transplant Outcomes Group enrolled between 2002 and 2010. Our primary outcome was Grade 3 PGD on Day 2 or 3. We measured preoperative plasma levels of five biomarkers (CC-16, sRAGE, ICAM-1, IL-8 and Protein C) that were previously associated with PGD when measured at the postoperative time point. We used multivariable logistic regression to adjust for potential confounders. Of 714 subjects, 130 (18%) developed PGD. Median CC-16 levels were elevated in subjects with PGD (10.1 vs. 6.0, p<0.001). CC-16 was associated with PGD in nonidiopathic pulmonary fibrosis (non-IPF) subjects (OR for highest quartile of CC-16: 2.87, 95% CI: 1.37, 6.00, p=0.005) but not in subjects with IPF (OR 1.38, 95% CI: 0.43, 4.45, p=0.59). After adjustment, preoperative CC-16 levels remained associated with PGD (OR: 3.03, 95% CI: 1.26, 7.30, p=0.013) in non-IPF subjects. Our study suggests the importance of preexisting airway epithelial injury in PGD. Markers of airway epithelial injury may be helpful in pretransplant risk stratification in specific recipients.

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.

Immunobiology of chronic lung allograft dysfunction: new insights from the bench and beyond.

The first successful human lung transplants were performed in the 1980s. Since that time lung transplantation has been a therapeutic modality for end-stage pulmonary diseases. However, chronic rejection, known as obliterative bronchiolitis (OB)/bronchiolitis obliterans syndrome (BOS), is the key reason why the 5-year survival is only 50%, which is significantly worse than most other solid organ transplants. Recent studies have provided exciting advances that are beginning to be translated into findings in humans. This review will highlight the current advances in understanding the mechanisms of OB/BOS in lung transplant recipients.

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.

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.

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.

B-lymphocytes in the lung: a topic to be revisited.

Humoral immunity is crucial to the immunologic homeostasis of the lung. Although having key roles in the clearance of infectious pathogens, humoral responses under certain condition may contribute to pathology in the lung. The regulation of local humoral immunity involves a highly complex network of antigen presenting cells, T and B-lymphocytes, as well as many membrane-bound and soluble signals. This review discusses B-lymphocyte function and immunoglogulin production in general, as well as the regulation and function of humoral immunity as it relates to the lung in health and disease.

Sarcoid myelopathy.

A 45-year-old woman with history of iritis, uveitis, and sarcoidosis of the skin presented with a subacute cervical myelopathy. Magnetic resonance imaging (MRI) showed patchy, multifocal, gadolinium-enhancing intramedullary lesions of the spinal cord, and extramedullary lesions of the basal cisterns and fourth ventricle. Symptoms and MRI abnormalities were improved within 1 month of corticosteroid therapy.

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.

Lung transplant metalloproteinase levels are elevated prior to bronchiolitis obliterans syndrome.

Parenchymal disease in the allograft lung is associated with interstitial remodeling believed to be mediated by matrix metalloproteinases (MMPs). Recent studies suggest high levels of MMP-9 are associated with bronchiolitis obliterans syndrome (BOS) in lung transplant recipients. Since BOS occurs late in the posttransplant period and may be preceded by episodes of acute rejection or infection, which are associated with interstitial remodeling, we examined MMP profiles in allograft bronchoalveolar lavage (BAL) fluid in the early posttransplant period (preceding BOS). Gelatin zymography, protein array analysis and specific ELISA on BAL fluids from transplanted lungs indicated that MMP-8, MMP-9 and TIMP-1 were strongly expressed in allograft BAL fluid from stable patients, or those with infection or rejection compared to BAL fluid from normal volunteers. Elevated expression of MMP-8, MMP-9 and TIMP-1 occurred early, and was sustained for the 3.2 years covered in this study. Elevations of MMP-8, MMP-9 and TIMP-1 in the first 2 years posttransplant appear to be associated with lung transplantation itself, and not infection or rejection. These data suggest that ongoing and clinically silent MMP activity could perpetuate progressive disease in the allograft lung.

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.

Alloantigen-induced immunoglobulin production in human lung: differential effects of accessory cell populations on IgG synthesis.

Local immunoglobulin production has been implicated in the pathogenesis of lung allograft rejection. The role of varying classes of lung accessory cells in stimulating an immunoglobulin (Ig) response in this setting as well as cytokines necessary for Ig synthesis is unknown. The purpose of the current study was to compare the accessory cell capabilities of lung dendritic cells (DC), parenchymal macrophages (PM), and alveolar macrophages (AM) in the generation of a humoral response to alloantigen. Allogeneic AM induced a dose-dependent production of IgG from peripheral blood mononuclear cells. In contrast, allogeneic DC and PM were unable to induce IgG synthesis. The inability of DC to stimulate IgG synthesis was observed despite a potent induction of T-cell proliferation and interferon-gamma (IFN-gamma) production. Additionally, supernatants from DC cultures suppressed AM-induced IgG production, suggesting that a soluble inhibitor of IgG synthesis was produced by DC-stimulated lymphocytes. AM-induced IgG synthesis was predominantly the result of IgG1 and IgG2 production. Experiments with blocking antibodies to either IFN-gamma or interleukin-4 (IL-4) revealed that both IFN-gamma and IL-4 participated in IgG synthesis, while only IFN-gamma was required for IgG2 production. These data demonstrate a discordance between the ability of lung accessory cells to induce T-cell proliferation and IgG synthesis. Furthermore, these findings suggest that local induction of either IL-4 or IFN-gamma is involved in stimulation of an IgG response to lung alloantigen.

Bronchoscopy in the diagnosis of respiratory infections.

In recent years, several factors have altered the spectrum of respiratory infections and their likelihood of response to empiric treatment. Altered microbial resistance has led to the possible need for specific etiologic diagnosis in some hospital-acquired infections in the normal host. In the immune-compromised host, the spectrum of atypical presentations and unusual organisms limits the clinician's ability to choose effective empiric therapies. In the normal host, bronchoscopic diagnosis seems to be most useful in the groups with severe community-acquired pneumonia or poor response to therapy for community-acquired pneumonia. The group of patients with ventilator-associated pneumonia has been well-researched and the bronchoscopic techniques tend to show increased sensitivity over other diagnostic means, but this has not been proven to alter morbidity, mortality, or cost effectiveness. The immune-compromised host is commonly infected by organisms not easily diagnosed by other means and is thus unable to be treated empirically. Bronchoscopic diagnostic techniques play a larger and more clearly delineated role in these populations, including the patient populations with solid organ transplants, bone marrow transplants, and AIDS.

Measurement of IL-6 inhibitory activity in cultured cell supernatants and lysates.

Interleukin-6 (IL-6) participates in a variety of cellular activities including regulation of immune and inflammatory responses. We have previously reported a discrepancy between bioactive and antigenic IL-6 secretion by lipopolysaccharide-stimulated alveolar macrophages (AMs) from smokers and have speculated that this may be due to cosecretion of an IL-6 inhibitor. In this study we further define our methods for measuring IL-6 inhibitory activity by testing the ability of serially diluted, cultured cell supernatants and lysates to suppress proliferation of an IL-6-dependent cell line, B9, to optimal concentrations of rIL-6. AM secretion of the inhibitory factor was optimal when AMs were stimulated with 1 micrograms/ml lipopolysaccharide (LPS). AMs from smokers secreted significantly greater amounts of this factor than AMs from nonsmokers. It was crucial to remove IL-6 from test samples on an IL-6 immunoaffinity column before analyzing for IL-6 inhibitory activity because (1) B9 cell proliferation could be suppressed by excess amounts of IL-6 in test supernatants and (2) excess rIL-6 added to the inhibitor assay reduced inhibitory activity. The latter finding suggested that IL-6 inhibitory activity was due to a competitive inhibitor of IL-6. This factor was shown to be specific for IL-6, because no inhibitory activity was seen on IL-2- or IL-4-dependent cell lines. Finally, we demonstrated that monocytes could also secrete an inhibitor of IL-6 bioactivity. However, secretion appeared to be less than that observed by AMs, suggesting that differentiation of monocytes into macrophages upregulated production of this factor.