The Prognostic Importance of Bronchoalveolar Lavage Fluid CXCL9 During Minimal Acute Rejection on the Risk of Chronic Lung Allograft Dysfunction.

The clinical significance and treatment strategies for minimal acute rejection (grade A1), the most common form of acute rejection (AR), remain controversial. In this retrospective single-center cohort study of 441 lung transplant recipients, we formally evaluate the association between minimal AR and chronic lung allograft dysfunction (CLAD) and test a novel hypothesis using bronchoalveolar lavage (BAL) CXCL9 concentration during minimal AR as a biomarker of subsequent CLAD development. In univariable and multivariable models adjusted for all histopathologic injury patterns, minimal AR was not associated with CLAD development. However, minimal AR with elevated BAL CXCL9 concentrations markedly increased CLAD risk in a dose-response manner. Minimal AR with CXCL9 concentrations greater than the 25th, 50th, and 75th percentile had adjusted hazard ratios (HRs) for CLAD of 1.1 (95% confidence interval [CI] 0.8-1.6), 1.6 (95% CI 1.1-2.3), and 2.2 (95% CI 1.4-3.4), respectively. Thus we demonstrate the utility of BAL CXCL9 measurement as a prognostic biomarker that allows discrimination of recipients at increased risk of CLAD development after minimal AR. BAL CXCL9 measurement during transbronchial biopsies may provide clinically useful prognostic data and guide treatment decisions for this common form of AR, as a possible strategy to minimize CLAD development.

Impact of Allograft Injury Time of Onset on the Development of Chronic Lung Allograft Dysfunction After Lung Transplantation.

The impact of allograft injury time of onset on the risk of chronic lung allograft dysfunction (CLAD) remains unknown. We hypothesized that episodes of late-onset (≥6 months) allograft injury would produce an augmented CXCR3/ligand immune response, leading to increased CLAD. In a retrospective single-center study, 1894 transbronchial biopsy samples from 441 lung transplant recipients were reviewed for the presence of acute rejection (AR), lymphocytic bronchiolitis (LB), diffuse alveolar damage (DAD), and organizing pneumonia (OP). The association between the time of onset of each injury pattern and CLAD was assessed by using multivariable Cox models with time-dependent covariates. Bronchoalveolar lavage (BAL) CXCR3 ligand concentrations were compared between early- and late-onset injury patterns using linear mixed-effects models. Late-onset DAD and OP were strongly associated with CLAD: adjusted hazard ratio 2.8 (95% confidence interval 1.5-5.3) and 2.0 (1.1-3.4), respectively. The early-onset form of these injury patterns did not increase CLAD risk. Late-onset LB and acute rejection (AR) predicted CLAD in univariable models but lost significance after multivariable adjustment for late DAD and OP. AR was the only early-onset injury pattern associated with CLAD development. Elevated BAL CXCR3 ligand concentrations during late-onset allograft injury parallel the increase in CLAD risk and support our hypothesis that late allograft injuries result in a more profound CXCR3/ligand immune response.

The Role of TGF-ß in the Association Between Primary Graft Dysfunction and Bronchiolitis Obliterans Syndrome.

Primary graft dysfunction (PGD) is a possible risk factor for bronchiolitis obliterans syndrome (BOS) following lung transplantation; however, the mechanism for any such association is poorly understood. Based on the association of TGF-ß with acute and chronic inflammatory disorders, we hypothesized that it might play a role in the continuum between PGD and BOS. Thus, the association between PGD and BOS was assessed in a single-center cohort of lung transplant recipients. Bronchoalveolar lavage fluid concentrations of TGF-ß and procollagen collected within 24 h of transplantation were compared across the spectrum of PGD, and incorporated into Cox models of BOS. Immunohistochemistry localized expression of TGF-ß and its receptor in early lung biopsies posttransplant. We found an association between PGD and BOS in both bilateral and single lung recipients with a hazard ratio of 3.07 (95% CI 1.76-5.38) for the most severe form of PGD. TGF-ß and procollagen concentrations were elevated during PGD (p < 0.01), and associated with increased rates of BOS. Expression of TGF-ß and its receptor localized to allograft infiltrating mononuclear and stromal cells, and the airway epithelium. These findings validate the association between PGD and the subsequent development of BOS, and suggest that this association may be mediated by receptor/TGF-ß biology.

Staphylococcus via an interaction with the ELR+ CXC chemokine ENA-78 is associated with BOS.

Staphylococcus aureus is the most commonly isolated gram-positive bacterium after lung transplantation (LT) and has been associated with poor posttransplant outcomes, but its effect on bronchiolitis obliterans syndrome (BOS) and death in the context of the allograft inflammatory environment has not been studied. A three-state Cox semi-Markovian model was used to determine the influence of allograft S. aureus and the ELR+ CXC chemokines on the survival rates and cause-specific hazards for movement from lung transplant (State 1) to BOS (State 2), from transplant (State 1) to death (State 3), and from BOS (State 2) to death (State 3). Acute rejection, pseudomonas pneumonia, bronchoalveolar lavage fluid (BALF) CXCL5 and its interaction with S. aureus all increased the likelihood of transition from transplant to BOS. Transition to death from transplant was facilitated by pseudomonas infection and single lung transplant. Movement from BOS to death was affected by the interaction between aspergillus, pseudomonas and CXCL5, but not S. aureus. S. aureus isolation had state specific effects after LT and only in concert with elevated BALF CXCL5 concentrations did it augment the risk of BOS. Pseudomonas and elevated BALF concentrations of CXCL5 continued as significant risk factors for BOS and death after BOS in lung transplantation.

Interstitial lung disease in patients with hepatopulmonary syndrome: a case series and new observations.

Hepatopulmonary syndrome (HPS) is characterized by impaired oxygenation due to pulmonary vascular dilatation in patients with end-stage liver disease. At our center, we identified 29 patients who were listed for liver transplantation (LT) with a model for end-stage liver disease exception for HPS between 2001 and 2012. Five of these patients were found to have concurrent interstitial lung disease (ILD). The chest high-resolution computed-tomography demonstrated ground-glass opacities and subpleural reticulation, most consistent with nonspecific interstitial pneumonia (NSIP). All four of our patients who underwent LT experienced prolonged hypoxemia postoperatively, with one surgery-related death. However, the three surviving patients had eventual resolution of their hypoxemia with no evidence of ILD progression. In conclusion, we report a high prevalence of ILD, most consistent with NSIP, among patients with HPS. Although there may be increased perioperative risks, the finding of ILD in patients with HPS should not be considered an absolute contraindication to LT.

Colonization with small conidia Aspergillus species is associated with bronchiolitis obliterans syndrome: a two-center validation study.

Aspergillus colonization after lung transplantation may increase the risk for bronchiolitis obliterans syndrome (BOS), a disease of small airways. We hypothesized that colonization with small conidia Aspergillus species would be associated with a greater risk of BOS, based upon an increased likelihood of deposition in small airways. We studied adult primary lung recipients from two large centers; 298 recipients at University of California, Los Angeles and 482 recipients at Duke University Medical Center. We grouped Aspergillus species by conidia diameter≤3.5 µm. We assessed the relationship of colonization with outcomes in Cox models. Pre-BOS colonization with small conidia Aspergillus species, but not large, was a risk factor for BOS (p=0.002, HR 1.44, 95% CI 1.14-1.82), along with acute rejection, single lung and Pseudomonas. Colonization with small conidia species also associated with risk of death (p=0.03, HR 1.30, 95% CI 1.03-1.64). Although other virulence traits besides conidia size may be important, we have demonstrated in two large independent cohorts that colonization with small conidia Aspergillus species increases the risk of BOS and death. Prospective evaluation of strategies to prevent Aspergillus colonization of small airways is warranted, with the goal of preserving lung allograft function as long as possible.

CXCR3 chemokine ligands during respiratory viral infections predict lung allograft dysfunction.

Community-acquired respiratory viruses (CARV) can accelerate the development of lung allograft dysfunction, but the immunologic mechanisms are poorly understood. The chemokine receptor CXCR3 and its chemokine ligands, CXCL9, CXCL10 and CXCL11 have roles in the immune response to viruses and in the pathogenesis of bronchiolitis obliterans syndrome, the predominant manifestation of chronic lung allograft rejection. We explored the impact of CARV infection on CXCR3/ligand biology and explored the use of CXCR3 chemokines as biomarkers for subsequent lung allograft dysfunction. Seventeen lung transplant recipients with CARV infection had bronchoalveolar lavage fluid (BALF) available for analysis. For comparison, we included 34 BALF specimens (2 for each CARV case) that were negative for infection and collected at a duration posttransplant similar to a CARV case. The concentration of each CXCR3 chemokine was increased during CARV infection. Among CARV infected patients, a high BALF concentration of either CXCL10 or CXCL11 was predictive of a greater decline in forced expiratory volume in 1 s, 6 months later. CXCR3 chemokine concentrations provide prognostic information and this may have important implications for the development of novel treatment strategies to modify outcomes after CARV infection.

Elevated plasma long pentraxin-3 levels and primary graft dysfunction after lung transplantation for idiopathic pulmonary fibrosis.

Primary graft dysfunction (PGD) after lung transplantation may result from ischemia reperfusion injury (IRI). The innate immune response to IRI may be mediated by Toll-like receptor and IL-1-induced long pentraxin-3 (PTX3) release. We hypothesized that elevated PTX3 levels were associated with PGD. We performed a nested case control study of lung transplant recipients with idiopathic pulmonary fibrosis (IPF) or chronic obstructive pulmonary disease (COPD) from the Lung Transplant Outcomes Group cohort. PTX3 levels were measured pretransplant, and 6 and 24 h postreperfusion. Cases were subjects with grade 3 PGD within 72 h of transplantation and controls were those without grade 3 PGD. Generalized estimating equations and multivariable logistic regression were used for analysis. We selected 40 PGD cases and 79 non-PGD controls. Plasma PTX3 level was associated with PGD in IPF but not COPD recipients (p for interaction < 0.03). Among patients with IPF, PTX3 levels at 6 and 24 h were associated with PGD (OR = 1.6, p = 0.02 at 6 h; OR = 1.4, p = 0.008 at 24 h). Elevated PTX3 levels were associated with the development of PGD after lung transplantation in IPF patients. Future studies evaluating the role of innate immune activation in IPF and PGD are warranted.

Systemic sclerosis and bilateral lung transplantation: a single centre experience.

Lung involvement is the leading cause of death in systemic sclerosis (SSc), but lung transplantation (LT) for systemic disease remains controversial. Our objective was to comprehensively evaluate post-LT outcomes for SSc compared to idiopathic pulmonary fibrosis (IPF). We retrospectively evaluated bilateral LT recipients (LTRs) with SSc or IPF at our centre between January 1, 2003 and December 31, 2007. The primary end-point was all-cause mortality at 1 yr post-LT. Secondary end-points included assessments of acute rejection (AR), pulmonary function, infection and chronic rejection. 14 patients with SSc and 38 patients with IPF underwent LT. Apart from a younger SSc cohort (53.2 versus 58.8 yrs; p = 0.02), the two groups were well matched. 1-yr all-cause mortality was no different between SSc (6.6%) and IPF (13.1%) groups, after adjusting for age (p = 0.62). Rates of (AR) ≥2 were significantly increased for the SSc compared with the IPF group (hazard ratio (HR) 2.91; p = 0.007). Other end-points, including chronic rejection, infection and pulmonary function, showed no differences. SSc LTRs experience similar survival 1 yr post-LT when compared to IPF. AR rates may be significantly higher in the SSc group. Longer follow-up is necessary to determine the effects of gastrointestinal dysfunction and AR on late allograft function in SSc LTR.

Periostitis secondary to prolonged voriconazole therapy in lung transplant recipients.

We report five cases of possible drug-induced periostitis associated with long-term use of voriconazole therapy after lung transplantation (LT). The diagnosis of periostitis was made by the documentation of bone pain, elevation of serum alkaline phosphatase and characteristic findings on radionuclide bone imaging in the absence of any identifiable rheumatologic disease. This periostitis appears similar to hypertrophic osteoarthopathy (HOA) but does not meet all criteria for HOA. In all patients, the symptoms resolved rapidly after discontinuation of voriconazole therapy. Awareness of this potential syndrome, which manifests as bone pain, elevated serum alkaline phosphatase and a bone scan suggestive of periostitis, is necessary in LT recipients on long-term voriconazole.

Spatiotemporal expression of chemokines and chemokine receptors in experimental anti-myeloperoxidase antibody-mediated glomerulonephritis.

Myeloperoxidase (MPO)-anti-neutrophil cytoplasmic autoantibody (ANCA)-associated necrotizing crescentic glomerulonephritis (NCGN) is characterized by abundant leucocyte infiltration. Chemokines are chemotactic cytokines involved in receptor-mediated recruitment of leucocytes. Our objective was to analyse spatiotemporal gene expression of chemokines and chemokine receptors in anti-MPO-mediated NCGN, to find potential targets for intervening with leucocyte influx. NCGN was induced in mice by co-administration of anti-MPO immunoglobulin (Ig)G and lipopolysaccharide. mRNA expression levels of chemokines and chemokine receptors were analysed in whole kidney lysates as well as in laser microdissected glomeruli and tubulo-interstitial tissue 1 and 7 day(s) after NCGN induction. Several chemokines and chemokine receptors were induced or up-regulated in anti-MPO-mediated NCGN, both on day 1 (chemokines CCL3, 5; CXCL2, 5, 13; receptor CXCR2) and on day 7 (chemokines CCL2, 5, 7, 8, 17, 20; CXCL1, 2, 5, 10; CX(3)CL1; receptors CCR2, 8; CX(3)CR1). The expression levels of most chemokines and receptors were higher in glomeruli than in the tubulo-interstitium. Because of the temporal induction of CXCR2 on day 1, we hypothesized CXCR2 as a potential target for treatment in anti-MPO-induced NCGN. Inhibition of CXCR2 using a goat-anti-CXCR2 serum prior to NCGN induction increased glomerular neutrophil influx but did not affect crescent formation and albuminuria. In conclusion, expression levels of various chemokines and chemokine receptors were increased in anti-MPO NCGN, and expressed particularly in glomeruli. These chemokines and receptors may serve as potential targets for treatment. Inhibition of a single target, CXCR2, did not attenuate anti-MPO NCGN. Combinatorial interventions may be necessary to avoid redundancy.

Aspergillus colonization of the lung allograft is a risk factor for bronchiolitis obliterans syndrome.

Multiple infections have been linked with the development of bronchiolitis obliterans syndrome (BOS) post-lung transplantation. Lung allograft airway colonization by Aspergillus species is common among lung transplant recipients. We hypothesized that Aspergillus colonization may promote the development of BOS and may decrease survival post-lung transplantation. We reviewed all lung transplant recipients transplanted in our center between January 2000 and June 2006. Bronchoscopy was performed according to a surveillance protocol and when clinically indicated. Aspergillus colonization was defined as a positive culture from bronchoalveolar lavage or two sputum cultures positive for the same Aspergillus species, in the absence of invasive pulmonary Aspergillosis. We found that Aspergillus colonization was strongly associated with BOS and BOS related mortality in Cox regression analyses. Aspergillus colonization typically preceded the development of BOS by a median of 261 days (95% CI 87-520). Furthermore, in a multivariate Cox regression model, Aspergillus colonization was a distinct risk factor for BOS, independent of acute rejection. These data suggest a potential causative role for Aspergillus colonization in the development of BOS post-lung transplantation and raise the possibility that strategies aimed to prevent Aspergillus colonization may help delay or reduce the incidence of BOS.

CXCR3 ligands are augmented during the pathogenesis of pulmonary sarcoidosis.

We and other investigators have hypothesised that the CXC chemokine receptor (CXCR)3/CXCR3 ligand biological axis is involved in the formation of sarcoid lung granulomas; however, significant discrepancies in the current literature remain. In an effort to clarify previous conflicting findings, we performed the largest observational study to date of interferon-inducible ELR(-) (lacking the sequence glutamic acid-leucine-arginine) CXC chemokines in sarcoid bronchoalveolar fluid (BALF). BALF chemokine levels from sarcoid patients (n = 72) and healthy controls (n = 8) were measured with the ELISA method. Immunohistochemical staining was performed for CXCR3 and its ligands. BALF CXC chemokine ligand (CXCL)10 levels from sarcoid patients were not significantly increased compared with controls. BALF CXCL11 levels from sarcoid patients demonstrated a trend towards elevation; subgroup analysis by stage showed significant BALF CXCL11 elevation in stage I sarcoid patients compared with controls. BALF CXCL9 levels were elevated from sarcoid patients compared with controls. CXC11, CXCL9 and CXCR3 were expressed from epithelioid histiocytes, multinucleated giant cells and other inflammatory cells forming sarcoid lung granulomas. Our data suggest that CXCL9 and CXCL11 are important mediators in recruiting CXCR3-expressing cells. Importantly, we have made the novel observation that both lymphocytes and cells of monocyte linage express CXCR3 and are involved in the formation of sarcoid lung granulomas.

Pulmonary hypertension associated with lung transplantation obliterative bronchiolitis and vascular remodeling of the allograft.

Pathologic obliterative bronchiolitis (OB)/Bronchiolitis obliterans syndrome (pathologic OB/BOS) is the major obstacle to long-term survival post-lung transplantation (LT). Our group has demonstrated that pulmonary hypertension (PH) complicates the course of chronic inflammatory lung diseases that have similarities to pathologic OB/BOS and that vascular remodeling of the bronchial circulation occurs during BOS. Consequently, we hypothesized that PH is associated with pathologic OB/BOS and may result from a vasculopathy of the allograft pulmonary circulation. We conducted a single-center, retrospective study and examined the presence of PH and vasculopathy in patients with pathologic OB/BOS. Fifty-two pathologic specimens post-LT were recovered from January 10, 1997 to January 5, 2007 and divided into two groups, those with and without pathologic OB/BOS.PH was defined as a mean pulmonary artery pressure (mPAP) > 25 mmHg by right heart catheterization (RHC) or right ventricular systolic pressure (RVSP) > or = 45 mmHg by transthoracic echocardiogram (TTE). PH was more prevalent in those LT recipients with pathologic OB/BOS (72% vs. 0%, p = 0.003). Furthermore, pulmonary arteriopathy and venopathy were more prevalent in patients with pathologic OB/BOS (84% vs. 4%, p < 0.0001, and 77% vs. 35%, p = 0.004, respectively). PH is common in LT recipients with pathologic OB/BOS and is associated with a vasculopathy of the allograft pulmonary circulation.

Altered levels of CC chemokines during pulmonary CMV predict BOS and mortality post-lung transplantation.

Pulmonary CMV infection (CMVI) and disease (CMVD) is associated with reduced long-term survival post-lung transplantation, however, the specific biologic mechanisms remain unclear. We have demonstrated a role of CC chemokines during lung allograft dysfunction. Based on these findings, we hypothesized that pulmonary CMV upregulates the expression of multiple CC chemokines that leads to allograft dysfunction and decreased long-term survival. We performed a nested case control study in lung transplant recipients to investigate alterations in CC chemokine biology during pulmonary CMV. Levels of CC chemokines were measured in bronchoalveolar lavage fluid (BALF) from recipients with CMVI (n = 33), CMVD (n = 6), and in healthy lung transplant controls (n = 33). We found a trend toward increased levels of MIP-1alpha/CCL3 during pulmonary CMVI. Levels of MCP-1/CCL2 and RANTES/CCL5 were significantly elevated during pulmonary CMV. Interestingly, elevated levels of CCL3 in BALF were protective with regards to survival. Importantly, elevated levels of CCL2 in BALF predicted the development of BOS, while elevated levels of CCL5 in BALF predicted an increase in mortality post-lung transplant. Altered levels of specific CC chemokines during pulmonary CMV are associated with future clinical outcomes. These results suggest a possible utility of BALF CC chemokines as biomarkers for guiding risk assessment during pulmonary CMV post-lung transplantation.

ENA-78 is an important angiogenic factor in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and often fatal disorder. Fibroplasia and deposition of extracellular matrix are dependent, in part, on angiogenesis and vascular remodeling. We obtained open lung biopsies from patients undergoing thoracic surgery for reasons other than interstitial lung disease (control) (n = 78) and from patients with IPF (n = 91). We found that levels of epithelial neutrophil-activating peptide 78 (ENA-78) were greater from tissue specimens of IPF patients, as compared with control subjects. When ENA-78 was depleted from IPF tissue specimens, tissue-derived angiogenic activity was markedly reduced. Immunolocalization of ENA-78 demonstrated that hyperplastic Type II pneumocytes and macrophages were the predominant cellular sources of ENA-78. These findings support the notion that ENA-78 may be an important additional factor that regulates angiogenic activity in IPF.

Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival after lung transplantation. Acute rejection, its main risk factor, is characterized by perivascular/bronchiolar leukocyte infiltration. BOS is characterized by persistent peribronchiolar leukocyte recruitment leading to airway fibrosis and obliteration. The specific mechanism(s) by which these leukocytes are recruited are unknown. Because MCP-1, acting through its receptor CCR2, is a potent mononuclear cell chemoattractant, we hypothesized that expression of this chemokine during an allogeneic-response promotes persistent recruitment of leukocytes and, ultimately, rejection. We found that elevated levels of biologically active MCP-1 in human bronchial lavage fluid (BALF) were associated with the continuum from acute to chronic allograft rejection. Translational studies in a murine model of BOS demonstrated increased MCP-1 expression paralleling mononuclear cell recruitment and CCR2 expression. Loss of MCP-1/CCR2 signaling, as seen in CCR2(-/-) mice or in WT mice treated with neutralizing antibodies to MCP-1, significantly reduced recruitment of mononuclear phagocytes following tracheal transplantation and led to attenuation of BOS. Lymphocyte infiltration was not reduced under these conditions. We suggest that MCP-1/CCR2 signaling plays an important role in recruitment of mononuclear phagocytes, a pivotal event in the pathogenesis of BOS.

IL-12 attenuates bleomycin-induced pulmonary fibrosis.

Interleukin (IL)-12 is a potent inducer of interferon (IFN)-gamma. We postulated that IL-12 would attenuate bleomycin-induced pulmonary fibrosis. To test this hypothesis, we administered IL-12 or murine serum albumin to bleomycin-treated mice by daily intraperitoneal injection until day 12. Mice treated with IL-12 demonstrated decreased hydroxyproline levels compared with control treated mice. Furthermore, administration of IL-12 led to a time-dependent increase in both lung and bronchoalveolar lavage fluid IFN-gamma. The antifibrotic effect of IL-12 could be attenuated with simultaneous administration of neutralizing anti-IFN-gamma antibodies. These findings support the notion that IL-12 attenuates bleomycin-induced pulmonary fibrosis via modulation of IFN-gamma production.

CXC chemokines in angiogenesis.

A variety of factors have been identified that regulate angiogenesis, including the CXC chemokine family. The CXC chemokines are a unique family of cytokines for their ability to behave in a disparate manner in the regulation of angiogenesis. CXC chemokines have four highly conserved cysteine amino acid residues, with the first two cysteine amino acid residues separated by one non-conserved amino acid residue (i.e., CXC). A second structural domain within this family determines their angiogenic potential. The NH2 terminus of the majority of the CXC chemokines contains three amino acid residues (Glu-Leu-Arg: the ELR motif), which precedes the first cysteine amino acid residue of the primary structure of these cytokines. Members that contain the ELR motif (ELR+) are potent promoters of angiogenesis. In contrast, members that are inducible by interferons and lack the ELR motif (ELR-) are potent inhibitors of angiogenesis. This difference in angiogenic activity may impact on the pathogenesis of a variety of disorders.