Holly L. Evanoff

Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis.

Cells within the synovial tissue may recruit mononuclear phagocytes into the synovial fluid and tissues of arthritic patients. We investigated the production of the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1) using sera, synovial fluid, synovial tissue, as well as macrophages and fibroblasts isolated from synovial tissues from 80 arthritic patients. MCP-1 levels were significantly higher (P less than 0.05) in synovial fluid from RA patients (mean 25.5 +/- 8.1 ng/ml [SE]) compared to synovial fluid from osteoarthritis (OA) patients (0.92 +/- 0.08), or from patients with other arthritides (2.9 +/- 1.5). MCP-1 levels in RA sera (8.44 +/- 2.33) were significantly greater than MCP-1 in normal sera (0.16 +/- 0.06). The quantities of RA synovial fluid IL-8, which is chemotactic for neutrophils and lymphocytes, and MCP-1 were strongly positively correlated (P less than 0.05). To examine the cellular source of MCP-1, RA synovial tissue macrophages and fibroblasts were isolated. Synovial tissue fibroblasts did not express MCP-1 mRNA, but could be induced to produce MCP-1 by stimulation with either IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), or LPS. In contrast, unlike normal peripheral blood monocytes or alveolar macrophages, RA synovial tissue macrophages constitutively expressed MCP-1 mRNA and antigen. Immunohistochemical analysis of synovial tissue showed that a significantly greater percentage of RA macrophages (50 +/- 8%) as compared to either OA macrophages (5 +/- 2) or normal macrophages (1 +/- 0.3) reacted with anti-MCP-1 antibodies. In addition, the synovial lining layer reacted with MCP-1 in both RA and OA synovial tissues. In contrast, only a minority of synovial fibroblasts (18 +/- 8%) from RA synovium were positive for immunolocalization of MCP-1. These results suggest that synovial production of MCP-1 may play an important role in the recruitment of mononuclear phagocytes during inflammation associated with RA and that synovial tissue macrophages are the dominant source of this cytokine.

Respiratory Syncytial Virus Predisposes Mice to Augmented Allergic Airway Responses Via IL-13-Mediated Mechanisms

The development of severe childhood asthma may be influenced by several factors including environmental and infectious stimuli. The causal relationship between infectious viral responses, such as respiratory syncytial virus (RSV), and severe asthma during early childhood is unclear. In these studies, the ability for an initial RSV infection to exacerbate and promote a more severe asthmatic-type response was investigated by combining established murine models of disease. We examined the ability of RSV to induce exacerbation of allergic disease over a relatively long period, leading to development of severe airway responses including airway inflammation and hyperreactivity. The preferential production of IL-13 during a primary RSV infection appears to play a critical role for the exacerbation of cockroach allergen-induced disease. The depletion of IL-13 during RSV infections inhibited the exacerbation and acceleration of severe allergen-induced airway hyperreactivity. This was indicated by decreases in airway hyperreactivity and changes in lung chemokine production. These data suggest that the airway responses during asthma can be greatly affected by a previous RSV infection, even when infection occurs before allergen sensitization. Overall, infection of the airways with RSV can induce an IL-13-dependent change in airway function and promotes an environment that contributes to the development of severe allergic asthmatic responses.

A Sensitive Elisa for the Detection of Human Monocyte Chemoattractant Protein-1 (MCP-1)

The recruitment of monocytes into tissue is associated with both acute and chronic inflammation. Although monocyte migration is measured in vitro by monocyte chemotaxis, this technique is often difficult to determine the specific quantitative contribution of a monocyte chemotaxin. We have developed a sensitive sandwich ELISA for the detection of monocyte chemoattractant protein-1 (MCP-1), a highly specific monocyte activating/chemotactic peptide. Polyclonal antibodies were generated from rabbits. The IgG fraction of the antiserum was isolated by a protein A column, with a portion of the antibodies biotinylated. Avidin-conjugated horse radish peroxidase was used for enzymatic, colorimetric analysis. The lower threshold for detection of MCP-1 was 50 pg/ml, and the ELISA was specific for MCP-1, since it failed to recognize other cytokines in a dose-dependent fashion. Furthermore, this ELISA had the capacity to measure endothelial cell and pulmonary fibroblast-derived MCP-1. The development of a sensitive ELISA for the detection MCP-1 is significant, since it will allow the measurement MCP-1 from biologically relevant fluids, and aid in establishing whether MCP-1 is present in disease.

Expression and Contribution of Endogenous IL-13 in an Experimental Model of Sepsis

IL-13 has been shown to exert potent anti-inflammatory properties. In this study, we elucidated the functional role of endogenous IL-13 in a murine model of septic peritonitis induced by cecal ligation and puncture (CLP). Initial studies demonstrated that the level of IL-13 increased in tissues including liver, lung, and kidney, whereas no considerable increase was found in either peritoneal fluid or serum after CLP. Immunohistochemically, IL-13-positive cells were Kupffer cells in liver, alveolar macrophages in lung, and epithelial cells of urinary tubules in kidney. IL-13 blockade with anti-IL-13 Abs significantly decreased the survival rate of mice after CLP from 53% to 14% on day 7 compared with control. To determine the potential mechanisms whereby IL-13 exerted a protective role in this model, the effects of anti-IL-13 Abs on both local and systemic inflammation were investigated. Administration of anti-IL-13 Abs did not alter the leukocyte infiltration and bacterial load in the peritoneum after CLP but dramatically increased the neutrophil influx in tissues after CLP, an effect that was accompanied by significant increases in the serum levels of aspartate transaminase, alanine transaminase, blood urea nitrogen, and creatinine. Tissue injury caused by IL-13 blockade was associated with increases in mRNA and the protein levels of CXC chemokines macrophage inflammatory protein-2 and KC as well as the CC chemokine macrophage inflammatory protein-1α and the proinflammatory cytokine TNF-α. Collectively, these results suggest that endogenous IL-13 protected mice from CLP-induced lethality by modulating inflammatory responses via suppression of overzealous production of inflammatory cytokines/chemokines in tissues.

Hyper-responsiveness of IPF/UIP fibroblasts: Interplay between TGFβ1, IL-13 and CCL2

One of the hallmarks of idiopathic pulmonary fibrosis with a usual interstitial pneumonia histological pathology (IPF/UIP) is excess collagen deposition, due to enhanced fibroblast extracellular matrix synthetic activity. Studies using murine models of lung fibrosis have elucidated a pro-fibrotic pathway involving IL-13 driving CCL2, which in turn drives TGFbeta1 in lung fibroblasts. Therefore, we sought to determine whether this pathway exists in the human fibrotic setting by evaluating human IPF/UIP fibroblasts. IPF/UIP fibroblasts have an increased baseline fibrotic phenotype compared to non-fibrotic fibroblasts. Interestingly, non-fibrotic fibroblasts responded in a pro-fibrotic manner to TGFbeta1 but were relatively non-responsive to IL-13 or CCL2, whereas, IPF/UIP cells were hyper-responsive to TGFbeta1, IL-13 and CCL2. Interestingly, TGFbeta1, CCL2 and IL-13 all upregulated TGFbeta receptor and IL-13 receptor expression, suggesting an ability of the mediators to modulate the function of each other. Furthermore, in vivo, neutralization of both JE and MCP5, the two functional orthologs of CCL2, during bleomycin-induced pulmonary fibrosis significantly reduced collagen deposition as well as JE and CCR2 expression. Also in the bleomycin model, CTGF, which is highly induced following TGFbeta stimulation, was attenuated with anti-JE/anti-MCP5 treatment. Overall this study demonstrates an interplay between TGFbeta1, IL-13 and CCL2 in IPF/UIP, where these three mediators feedback on each other, promoting the fibrotic response.

Pivotal Role of the CC Chemokine, Macrophage-Derived Chemokine, in the Innate Immune Response

Macrophage-derived chemokine (MDC), a recently identified CC chemokine, has been regarded to be involved in chronic inflammation and dendritic cell and lymphocyte homing. In this study, we demonstrate a pivotal role for MDC during experimental sepsis induced by cecal ligation and puncture (CLP). Intraperitoneal administration of MDC (1 μg/mouse) protected mice from CLP-induced lethality. The survival was accompanied by increased number of peritoneal macrophages and decreased recovery of viable bacteria from the peritoneum and peripheral blood. In addition, mice treated with an i.p. injection of MDC cleared bacteria more effectively than those in the control when 3 × 108 CFU live Escherichia coli was i.p. inoculated. Endogenous MDC was detected in the peritoneum after CLP, and neutralization of the MDC with anti-MDC Abs decreased CLP-induced recruitment of peritoneal macrophages and increased the recovery of viable bacteria from the peritoneum and peripheral blood. MDC blockade was deleterious in the survival of mice after CLP. In vitro, MDC enhanced the phagocytic and killing activities of peritoneal macrophages to E. coli and induced both a respiratory burst and the release of lysozomal enzyme from macrophages. Furthermore, MDC dramatically ameliorated CLP-induced systemic tissue inflammation as well as tissue dysfunction, which were associated in part with decreased levels of TNF-α, macrophage inflammatory proteins-1α and -2, and KC in specific tissues. Collectively, these results indicate novel regulatory activities of MDC in innate immunity during sepsis and suggest that MDC may aid in an adjunct therapy in sepsis.

TLR9 Differentiates Rapidly from Slowly Progressing Forms of Idiopathic Pulmonary Fibrosis

Compared to slow progressors, patients with rapidly progressive idiopathic pulmonary fibrosis express more TLR9, which recognizes unmethylated CpG DNA and stimulates the fibrotic process. Taking a Toll on Breathing Despite the incredible rate of advances being made in medical science, the exact causes of many diseases remain unknown. These diseases are classified as idiopathic—“a disease of its own kind.” But like a thief who leaves clues at a crime scene that disclose his or her identity, diseases can spur aberrant biological processes that hint at the condition’s cause. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive form of lung disease of unknown origin characterized by the excess production of fibrous connective tissue (fibrosis) in the supporting framework (interstitium) of the lungs. These changes cause the hardening and/or scarring of lung tissue due to excess collagen, resulting in shortness of breath, a chronic dry cough, fatigue, weakness, chest discomfort, loss of appetite, and rapid weight loss. Patients with IPF have a poor prognosis and are usually expected to live only an average of 4 to 6 years after diagnosis; however, IPF displays a very heterogeneous path, with disease progressing rapidly in some patients and more slowly in others. Thus far, physicians have been unable to predict the speed of disease progression in patients newly diagnosed with IPF. Now, Trujillo et al. have identified a marker that differentiates these two patient groups and that may also mediate rapid progression of this disease. Toll-like receptor 9 (TLR9) is an innate immune molecule that recognizes a particular type of DNA frequently found in bacteria and viruses—unmethylated CpG DNA. Signaling through TLR9 promotes the differentiation of lung fibroblasts taken from IPF patients into myofibroblasts—cells that resemble both smooth muscle and fibroblasts—a key process in fibrosis. Trujillo et al. hypothesized that TLR9 may contribute to rapidly progressing IPF. Indeed, they found higher amounts of TLR9 in rapidly progressing IPF patients compared to slow progressing patients and normal controls. Moreover, in a xenograft mouse model of IPF, fibroblasts from rapid progressors induced more severe fibrosis in response to TLR9 activation than those from slow progressors. The presence of CpG also induced epithelial to mesenchymal transition—another hallmark of fibrosis—in a lung epithelial cell line in vitro. Together, these results suggest that TLR9 may serve as a marker for IPF rapid progressors and that TLR9 targeting may be a new therapeutic strategy for treating IPF. Thus, although the cause(s) of IPF remains unknown, the new data offer hope for an improvement in the prognosis and possibly treatment of this devastating disease. Idiopathic pulmonary fibrosis is characterized by diffuse alveolar damage and severe fibrosis, resulting in a steady worsening of lung function and gas exchange. Because idiopathic pulmonary fibrosis is a generally progressive disorder with highly heterogeneous disease progression, we classified affected patients as either rapid or slow progressors over the first year of follow-up and then identified differences between the two groups to investigate the mechanism governing rapid progression. Previous work from our laboratory has demonstrated that Toll-like receptor 9 (TLR9), a pathogen recognition receptor that recognizes unmethylated CpG motifs in bacterial and viral DNA, promotes myofibroblast differentiation in lung fibroblasts cultured from biopsies of patients with idiopathic pulmonary fibrosis. Therefore, we hypothesized that TLR9 functions as both a sensor of pathogenic molecules and a profibrotic signal in rapidly progressive idiopathic pulmonary fibrosis. Indeed, TLR9 was present at higher concentrations in surgical lung biopsies from rapidly progressive patients than in tissue from slowly progressing patients. Moreover, fibroblasts from rapid progressors were more responsive to the TLR9 agonist, CpG DNA, than were fibroblasts from slowly progressing patients. Using a humanized severe combined immunodeficient mouse, we then demonstrated increased fibrosis in murine lungs receiving human lung fibroblasts from rapid progressors compared with mice receiving fibroblasts from slowly progressing patients. This fibrosis was exacerbated by intranasal CpG challenges. Furthermore, CpG induced the differentiation of blood monocytes into fibrocytes and the epithelial-to-mesenchymal transition of A549 lung epithelial cells. These data suggest that TLR9 may drive the pathogenesis of rapidly progressive idiopathic pulmonary fibrosis and may serve as a potential indicator for this subset of the disease.

Human Pulmonary Fibroblasts Exhibit Altered Interleukin-4 and Interleukin-13 Receptor Subunit Expression in Idiopathic Interstitial Pneumonia

Abnormal proliferation of pulmonary fibroblasts is a prominent feature of chronic pulmonary fibrotic diseases such as idiopathic interstitial pneumonia (IIP), but it is not presently clear how this proliferative response by lung fibroblasts can be therapeutically modulated. In the present study, we examined whether it was possible to selectively target primary human pulmonary fibroblasts grown out of surgical lung biopsies (SLBs) from IIP patients based on their expression of interleukin-4 receptor (IL-4R) and IL-13R subunits. Pulmonary fibroblast lines cultured from patients with the severest form of IIP, namely usual interstitial pneumonia, exhibited the greatest gene and protein expression of IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2 compared with primary pulmonary fibroblast lines grown from other IIP SLBs and normal SLBs. When exposed to increasing concentrations of a chimeric protein comprised of human IL-13 and a truncated version of Pseudomonas exotoxin (IL13-PE), the proliferation of primary usual interstitial pneumonia fibroblasts was inhibited to a much greater extent compared with fibroblast lines from nonspecific interstitial pneumonia and respiratory bronchiolitis/interstitial lung disease patient groups. Fibroblasts from normal patients exhibited minimal susceptibility to the cytotoxic effect of IL13-PE. IL13-PE-mediated targeting of IIP fibroblasts was dependent on their expression of IL-4Ralpha and IL-13Ralpha2. Thus, these data suggest that the abnormal proliferative properties of human lung fibroblasts from certain IIP patient groups can be modulated in a manner that is dependent on the IL-4 and IL-13 receptor subunit expression by these cells.

Mast cells produce ENA-78, which can function as a potent neutrophil chemoattractant during allergic airway inflammation.

The inflammatory response during allergic airway inflammation involves the recruitment of multiple leukocyte populations, including neutrophils, monocytes, lymphocytes, and eosinophils. All of these populations likely contribute to the pathology observed during repeated episodes of allergic airway inflammation. We have examined the role of a human neutrophil‐specific chemokine (C‐x‐C), ENA‐78, in a model of allergic airway responses and identified murine mast cells as a cellular source of an ENA‐78‐like molecule. Within this allergic airway model, neutrophil infiltration into the airway occurs within 4–8 h post‐allergen challenge, persists within the airway until 24 h, and resolves by 48 h post‐challenge. Neutrophil influx precedes the eosinophil infiltration, which peaks in the airway at 48 h post‐allergen challenge. In this study the production of ENA‐78 from challenged lungs demonstrated a significant increase in the allergen‐,but not vehicle‐, challenged lungs. In vivo neutralization of ENA‐78 by passive immunization demonstrated a significant decrease in peak neutrophil infiltration at 8 h, with no effect on the eosinophil infiltration at 48 h post‐challenge. Because ENA‐78 has been shown to be chemotactic for neutrophils and given the involvement of mast cell degranulation in allergic responses, we examined mast cells for the presence of ENA‐78. Cultured mast cells spontaneously released ENA‐78, but on activation with IgE + antigen, NG‐L‐arginine methyl ester or compound 48/80 produced significantly increased levels of ENA‐78. Supernatants from sonicated MC‐9 mast cells induced an overwhelming influx of neutrophils into the BAL by 4 h post‐intratracheal injection into mice, suggesting that the mast cell is a significant source of neutrophil chemotactic factors. Mast cell supernatant‐mediated neutrophil infiltration was substantially decreased by preincubation of the supernatant with antibodies specific for ENA‐78. These data indicate a major neutrophil chemotactic protein produced by mast cells during allergic responses may be mast cell‐derived ENA‐78. J. Leukoc. Biol. 63: 746–751; 1998.

Role of interleukin-12 and stat-4 in the regulation of airway inflammation and hyperreactivity in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a respiratory pathogen that can cause significant morbidity in infants and young children. Interestingly, the majority of children who acquire a RSV infection do not exhibit severe symptoms. Development of a Th1 response has been associated with resolution of symptoms in viral infections and may explain mild RSV illness. The current study investigated the cytokine response observed in mild disease in C57BL/6 mice that had low airway resistance and mucus production with little pulmonary inflammation. RSV infection in these mice was accompanied by a fourfold increase in interleukin-12(IL-12). Treatment of RSV-infected mice with anti-IL-12 resulted in an increase in airway hyperreactivity, mucus production, and airway inflammation (eosinophilia). Since IL-12 activation is dependent on Stat-4-mediated intracellular signal transduction, similar experiments were performed in Stat-4 deficient mice and demonstrated similar results to those obtained from anti-IL-12 treated mice. Again, there was an increase in airway hyperreactivity and mucus production, and goblet cell hypertrophy. These studies support the importance of IL-12 in the immune response to RSV infection resulting in resolution of disease and protection from inappropriate inflammatory responses.