David K. Madtes

A significant part of macrophage-derived growth factor consists of at least two forms of PDGF

The macrophage has been suggested to be responsible for the connective tissue cell proliferation that accompanies most chronic inflammatory responses. One of the secretory products of activated macrophages is MDGF, a growth factor (or factors) for fibroblasts, 3T3 cells, smooth muscle, and vascular endothelium. This report demonstrates that a significant portion of the mitogenic activity for 3T3 cells secreted by cultured human alveolar and peritoneal macrophages is due to a molecule (or molecules) similar to platelet-derived growth factor (PDGF). Two size classes (approximately 37,000-39,000 and 12,000-17,000 daltons) of mitogenically active PDGF-like molecules are detected by two criteria--antigenic similarity with PDGF and ability to compete with 125I-PDGF for high-affinity binding to the PDGF receptor. The presence of mRNA for the B chain of PDGF is demonstrated by Northern analysis, and de novo synthesis of these molecules by activated macrophages is shown by immunoprecipitation of 35S-labeled proteins with anti-PDGF IgG.

Induction of transforming growth factor-α in activated human alveolar macrophages

Abstract The early monocyte infiltration observed in normal wound repair and in a number of pathologic processes precedes the epithelial and connective tissue proliferative responses, suggesting that the monocyte/macrophage may be an important source of growth factors for these tissues. In culture, activated macrophages secrete growth factors active on fibroblasts, smooth muscle, endothelium, and epithelium. This report demonstrates that activated human alveolar macrophages express the gene for transforming growth factor-α (TGF-α) in an inducible manner and secrete a factor into the culture medium that is functionally and immunologically identical to TGF-α. Two different molecular species of TGF-α activity (approximately 8,500 – 12,000 and 28,500 daltons) are identified in macrophage-conditioned medium. These observations establish the macrophage as a diploid human cell capable of synthesizing and secreting TGF-α. The activated macrophage therefore represents a cellular source of a mitogenic factor that is potentially important in epithelial proliferation and repair.

Obstructive Lung Disease after Allogeneic Marrow Transplantation: Clinical Presentation and Course

To describe the clinical presentation and progression of obstructive lung disease after marrow transplantation, we examined a sequential sample of 35 patients who had allogeneic marrow transplantation between January 1980 and January 1987, were 16 years or older, had normal pulmonary function tests before transplantation, and developed airflow obstruction defined as FEV1/FVC less than 70% and FEV1 less than 80% predicted 50 days or more after transplantation. Cases were selected from 1029 adult (older than 16 years) patients who underwent allogeneic marrow transplantation during the same period. Patients with airflow obstruction presented with symptoms of cough, dyspnea, or wheezing, or a combination. In 80% the chest radiograph was normal. Airflow obstruction was diagnosed within 1.5 years after transplantation in 33 of 35 patients. Clinical, extensive, chronic graft-versus-host disease was present in 24 patients. Only 4 patients had a complete response to primary therapy of chronic graft-versus-host disease. Serum IgG and IgA levels were decreased in 15 and 25 patients, respectively. The FEV1 declined rapidly (decrease in FEV1 greater than 30% between tests) in 21 patients, but 14 patients with slowly progressive or reversible disease were identified. Mortality was 65% at 3 years after transplant, a significantly higher value (P = 0.016) than the 3-year mortality rate of 44% in a comparison group of 412 concurrent patients with chronic graft-versus-host disease who were 16 years or older, survived more than 80 days after transplantation, and had normal pulmonary function. We concluded that obstructive lung disease after marrow transplantation may be variable with respect to time of onset and rate of progression. Obstructive lung disease was frequently associated with serum immunoglobulin deficiency and clinical, extensive, chronic graft-versus-host disease that was not readily responsive to treatment. Mortality was high but long-term survivors were identified.

Pulmonary venoocclusive disease following bone marrow transplantation.

We report two cases of pulmonary venoocclusive disease (PVOD) in children with acute lymphoblastic leukemia treated by marrow allograft transplantation following conditioning with high-dose 1-3 bis chloroethyl-1 nitrosourea (BCNU), etoposide (VP-16), and cyclophosphamide (Cy). Both patients developed symptomatic pulmonary hypertension documented by right heart catheterization. Open-lung biopsy of one patient demonstrated PVOD evident even on frozen sections stained with hematoxylin and eosin. High-dose methylprednisolone was associated with significant clinical improvement in both patients. Pulmonary symptoms resolved in one patient who subsequently died in leukemic relapse. PVOD resolved in the other patient, only to recur when steroids were discontinued and then again respond to reinstitution of therapy. More aggressive therapy for malignant diseases may increase the incidence of PVOD. Prompt recognition of its subtle clinical and histological manifestations allows early institution of steroid therapy, which may be beneficial.

Idiopathic pneumonia after bone marrow transplantation: cytokine activation and lipopolysaccharide amplification in the bronchoalveolar compartment.

OBJECTIVE To determine whether idiopathic pneumonia syndrome (IPS), a form of noninfectious lung injury that follows bone marrow transplantation, is associated with cytokine activation and increased susceptibility to lipopolysaccharide (LPS). DESIGN Case series. SETTING Tertiary referral center for marrow transplantation. PATIENTS Recipients with biopsy-confirmed IPS; normal volunteers and marrow transplant recipients without IPS were analyzed as controls. MEASUREMENTS AND MAIN RESULTS Levels of lymphocyte and macrophage-derived cytokines as well as components of the LPS, LPS-binding protein (LBP), and CD14 system in bronchoalveolar lavage (BAL) fluid were determined. We found evidence of increased vascular permeability (BAL protein) and inflammatory cytokine activation (interleukin-1, interleukin-2, interleukin-6, and tumor necrosis factor-alpha) in patients with IPS. Patients without IPS had BAL fluid cytokine and protein levels that were similar to levels in BAL fluid from normal volunteers. Moreover, components of the LPS amplification system (LBP and soluble CD14) were increased in patients with IPS but not in patients without IPS. CONCLUSIONS These results provide direct evidence for proinflammatory cytokine activation in IPS and suggest that these patients might be at increased risk for LPS-mediated injury through the LBP amplification pathway.

Myeloperoxidase Inactivates TIMP-1 by Oxidizing Its N-terminal Cysteine Residue AN OXIDATIVE MECHANISM FOR REGULATING PROTEOLYSIS DURING INFLAMMATION

An imbalance between the proteolytic activity of matrix metalloproteinases (MMPs) and the activity of tissue inhibitors of metalloproteinases (TIMPs) is implicated in tissue injury during inflammation. The N-terminal cysteine of TIMP-1 plays a key role in the inhibitory activity of the protein because it coordinates the essential catalytic Zn2+ of the MMP, preventing the metal ion from functioning. An important mechanism for controlling the interaction of TIMPs with MMPs might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase (MPO) system of phagocytes. Here, we show that HOCl generated by the MPO-H2O2-chloride system inactivates TIMP-1 by oxidizing its N-terminal cysteine. The product is a novel 2-oxo acid. Liquid chromatography-mass spectrometry and tandem mass spectrometry analyses demonstrated that methionine and N-terminal cysteine residues were rapidly oxidized by MPO-derived HOCl but only oxidation of the N-terminal cysteine of TIMP-1 correlated well with loss of inhibitory activity. Importantly, we detected the signature 2-oxo-acid N-terminal peptide in tryptic digests of bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome, demonstrating that TIMP-1 oxidation occurs in vivo. Loss of the N-terminal amino group and disulfide structure are crucial for preventing TIMP-1 from inhibiting MMPs. Our findings suggest that pericellular production of HOCl by phagocytes is a pathogenic mechanism for impairing TIMP-1 activity during inflammation.

Immunolocalization of Transforming Growth Factor-α, Epidermal Growth Factor (EGF), and EGF-Receptor in Normal and Injured Developing Human Lung

ABSTRACT: The family of growth factors that includes epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) are thought to play a role in the regulation of fetal lung development and epithelial repair after injury. To further elucidate the potential role of these growth factors and their receptor in normal human lung development and in response to injury, their distribution was determined by immunohistochemistry in normal fetal lung, as well as both normal and injured postnatal human lung. We studied 14 specimens of human lung tissue: from three fetuses, four normal infants, two preterm infants with hyaline membrane disease, and five infants with late bronchopulmonary dysplasia (BPD). EGF, TGF-α, and EGF receptor (EGF-R) colocalized in airway epithelium in normal fetal and in postnatal human lung. They were also colocalized in scattered alveolar epithelial cells in postnatal lung. Large numbers of alveolar macrophages immunostained for EGF, TGF-α, and EGF-R in lungs with late stages of BPD. The colocalization of these growth factors suggests parallel expression of EGF family members. Moreover, the colocalization of these growth factors with their receptor in developing lung suggests that they may act through an autocrine mechanism. The prominent expression of these growth factors in alveolar macrophages in BPD suggests they may be involved with the pathogenesis of this disease.

Incidence, risk factors, and mortality from pneumonia developing late after hematopoietic stem cell transplantation

Summary:The incidence, etiology, outcome, and risk factors for developing pneumonia late after hematopoietic stem cell transplantation (SCT) were investigated in 1359 patients transplanted in Seattle. A total of 341 patients (25% of the cohort) developed at least one pneumonic episode. No microbial or tissue diagnosis (ie clinical pneumonia) was established in 197 patients (58% of first pneumonia cases). Among the remaining 144 patients, established etiologies included 33 viral (10%), 31 bacterial (9%), 25 idiopathic pneumonia syndrome (IPS, 7%), 20 multiple organisms (6%), 19 fungal (6%), and 16 Pneumocystis carinii pneumonia (PCP) (5%). The overall cumulative incidence of first pneumonia at 4 years after discharge home was 31%. The cumulative incidences of pneumonia according to donor type at 1 and 4 years after discharge home were 13 and 18% (autologous/syngeneic), 22 and 34% (HLA-matched related), and 26 and 39% (mismatched related/unrelated), respectively. Multivariate analysis of factors associated with development of late pneumonia after allografting were increasing patient age (RR 0.5 for <20 years, 1.2 for >40 years, P=0.009), donor HLA-mismatch (RR 1.6 for unrelated/mismatched related, P=0.01), and chronic graft-versus-host disease (GVHD; RR 1.5, P=0.007). Our data suggest that extension of PCP prophylaxis may be beneficial in high-risk autograft recipients. Further study of long-term anti-infective prophylaxis based on patient risk factors after SCT appear warranted.

IL-13 and IL-1β promote lung fibroblast growth through coordinated up-regulation of PDGF-AA and PDGF-Rα

Peribronchiolar fibrosis is a prominent feature of airway remodeling in asthma and involves fibroblast growth and collagen deposition. Interleukin‐13 (IL‐13), a T‐helper 2 cytokine, is a key mediator of airway remodeling in asthma, yet the mechanism through which IL‐13 promotes fibroblast growth has not been investigated. In this study, we show that IL‐13 stimulates the mitogenesis of mouse, rat, and human lung fibroblasts through release of a soluble mitogen that we identified as PDGF‐AA. The IL‐13‐induced growth of human lung fibroblasts was attenuated by an anti‐PDGF‐AA neutralizing antibody, and IL‐13 stimulated human lung fibroblasts to secrete PDGF‐AA. Fibroblasts derived from mouse embryos possessing the lethal Patch mutation, which lack the PDGF‐Rα, showed no mitogenic response to IL‐13. However, Patch cells did exhibit IL‐13‐induced STAT‐6 phosphorylation. Stable transfection of the PDGF‐Rα into Patch cells restored the growth response to PDGF‐AA and IL‐13. Through the use of lung fibroblasts from STAT‐6‐deficient mice, we showed that IL‐13‐induced PDGF‐AA release is STAT‐6 dependent, but PDGF‐AA‐induced growth is STAT‐6 independent. Finally, we showed that IL‐1β enhanced IL‐13‐induced mitogenesis of rat lung fibroblasts through up‐regulation of the PDGF‐Rα. Our findings indicate that IL‐13 acts in synergy with IL‐1β to stimulate growth by coordinately up‐regulating PDGF‐AA and the PDGF‐Rα, respectively.

Collagen accumulation is decreased in SPARC-null mice with bleomycin-induced pulmonary fibrosis

Secreted protein acidic and rich in cysteine (SPARC) has been shown to be coexpressed with type I collagen in tissues undergoing remodeling and wound repair. We speculated that SPARC is required for the accumulation of collagen in lung injury and that its absence would attenuate collagen accumulation. Accordingly, we have assessed levels of collagen in SPARC-null mice in an intratracheal bleomycin-injury model of pulmonary fibrosis. Eight- to ten-week-old SPARC-null and wild-type (WT) mice received bleomycin (0.0035 U/g) or saline intratracheally and were subsequently killed after 14 days. Relative levels of SPARC mRNA were increased 2.7-fold (P < 0.001) in bleomycin-treated WT lungs in comparison with saline-treated lungs. Protein from bleomycin-treated WT lung contained significantly more hydroxyproline (191.9 microg/lung) than protein from either bleomycin-treated SPARC-null lungs or saline-treated WT and SPARC-null lungs (147.4 microg/lung, 125.4 microg/lung, and 113. 0 microg/lung, respectively; P < 0.03). These results indicate that SPARC is increased in response to lung injury and that accumulation of collagen, as indicated by hydroxyproline content, is attenuated in the absence of SPARC. The properties of SPARC as a matricellular protein associated with cell proliferation and matrix turnover are consistent with its participation in the development of pulmonary fibrosis.Secreted protein acidic and rich in cysteine (SPARC) has been shown to be coexpressed with type I collagen in tissues undergoing remodeling and wound repair. We speculated that SPARC is required for the accumulation of collagen in lung injury and that its absence would attenuate collagen accumulation. Accordingly, we have assessed levels of collagen in SPARC-null mice in an intratracheal bleomycin-injury model of pulmonary fibrosis. Eight- to ten-week-old SPARC-null and wild-type (WT) mice received bleomycin (0.0035 U/g) or saline intratracheally and were subsequently killed after 14 days. Relative levels of SPARC mRNA were increased 2.7-fold ( P < 0.001) in bleomycin-treated WT lungs in comparison with saline-treated lungs. Protein from bleomycin-treated WT lung contained significantly more hydroxyproline (191.9 μg/lung) than protein from either bleomycin-treated SPARC-null lungs or saline-treated WT and SPARC-null lungs (147.4 μg/lung, 125.4 μg/lung, and 113.0 μg/lung, respectively; P < 0.03). These results indicate that SPARC is increased in response to lung injury and that accumulation of collagen, as indicated by hydroxyproline content, is attenuated in the absence of SPARC. The properties of SPARC as a matricellular protein associated with cell proliferation and matrix turnover are consistent with its participation in the development of pulmonary fibrosis.