Kevin E. Driscoll

Macrophage Inflammatory Proteins: Biology and Role in Pulmonary Inflammation

Macrophage inflammatory proteins 1 alpha and beta (MIP-1 alpha and beta) and macrophage inflammatory protein 2 (MIP-2) are approximately 6-8 kd, heparin binding proteins that exhibit a number of inflammatory and immunoregulatory activities. The MIP proteins are members of a superfamily of cytokines called chemokines, many of which have been shown to possess chemotactic activity for inflammatory and immune effector cells. While MIPs were originally identified as secretory products of endotoxin-stimulated mouse macrophages, these chemokines are produced by a variety of cell types including neutrophils, fibroblasts, and epithelial cells. In addition, proteins with a high degree of structural and functional homology to murine MIP-1 alpha and beta and MIP-2 have been identified in other species including humans. MIP-1 alpha and beta are chemotactic for monocytes and lymphocytes and MIP-2 is a potent chemotactic factor for neutrophils. MIPs likely also play a role in regulating hematopoiesis and stimulating production of other inflammatory mediators such as IL-1, TNF alpha, and histamine. Studies using animal models of lung injury and inflammation have implicated MIPs as important mediators of lung defense. Increased MIP expression has been observed in models of bacterial sepsis, silicosis, and oxidant-induced lung injury. Studies in humans indicate MIP-1 alpha contributes to the inflammatory cell response associated with sarcoidosis and idiopathic pulmonary fibrosis. Given the bioactivities of MIP-1 alpha and beta and MIP-2 and the recent studies demonstrating their association with lung inflammation, it is likely these chemokines play a significant role in respiratory tract defenses and may contribute to the pathogenesis of inflammatory lung disease.

TNFα and MIP-2 : role in particle-induced inflammation and regulation by oxidative stress

The cytokine tumor necrosis factor alpha (TNFalpha) plays a critical role in particle-induced inflammation in the lung. TNFalpha production by macrophage can be stimulated by a variety of noxious particles and initiate a cascade of responses involving adhesion molecule expression and production of chemotactic cytokines which ultimately result in the infiltration of inflammatory cells to site of infection or tissue injury in the respiratory tract. Regarding chemotactic cytokines, TNFalpha is a potent agonist of chemokine expression in both immune and non-immune cells (e.g. epithelial cells, fibroblasts). The chemokine macrophage inflammatory protein-2 (MIP-2) plays a major role in mediating the neutrophilic inflammatory response of the rodent lung to particles such as quartz, crocidolite asbestos, as well as high doses of other relative innocuous dusts such as titanium dioxide. The documented sources of MIP-2 in the rodent lung after particle exposure include macrophages as well as epithelial cells. Recent studies indicate that expression of the MIP-2 gene in rat lung epithelial cells is dependent on the transcription factor NFkappaB and is regulated, in part, by oxidative stress induced by particle exposure.

CLONING, EXPRESSION, AND FUNCTIONAL CHARACTERIZATION OF RAT MIP-2 : A NEUTROPHIL CHEMOATTRACTANT AND EPITHELIAL CELL MITOGEN

Macrophage inflammatory protein‐2 (MIP‐2) is a member of a family of cytokines that play roles in inflammatory, immune, and wound healing responses. To clone the cDNA for rat MIP‐2, RNA was isolated from the lungs of Fischer 344 rats after instillation of lipopolysaccharide. Reverse transcription‐polymerase chain reaction was performed by using synthetic oligonucleotide primers designed from the mouse MIP‐2 cDNA sequence. A cDNA containing the coding region of rat MIP‐2 was cloned and sequenced. Comparison to the mouse MIP‐2 cDNA demonstrated 90.3% homology at the nucleotide level and 86% homology at the amino acid level. The rat MIP‐2 cDNA was expressed in Escherichia coli and protein evaluated for bioactivity. The recombinant rat MIP‐2 was chemotactic for rat neutrophils but did not stimulate migration of rat alveolar macrophages or human peripheral blood eosinophils or lymphocytes. In addition, the recombinant rat MIP‐2 and the related rat chemokine, KC/CINC stimulated proliferation of rat alveolar epithelial cells but not fibroblasts in vitro.

The comparison of a fibrogenic and two nonfibrogenic dusts by bronchoalveolar lavage

Analysis of bronchoalveolar lavage fluid (BALF) appears to be a sensitive approach to characterizing an acute inflammatory response within the lung. More work, however, is needed to determine if analyses of BALF endpoints can predict chronic responses (i.e., fibrosis). The objective of the present study was to compare the dose and temporal pulmonary response of a known fibrogenic agent, silica, and two known nonfibrogenic agents, aluminum oxide and titanium dioxide. Animals were instilled with silica (0, 0.2, 1.0, or 5.0 mg/100 g body wt), titanium dioxide (1.0 or 5 mg/100 g body wt), aluminium oxide (1.0 or 5.0 mg/100 g body wt) or saline. Animals (n = 5/group) were terminated 1, 7, 14, 28, and 63 days following instillation, and the BALF was characterized by biochemical and cellular assays. Histopathological changes were determined at 60 days after exposure. The biochemical results demonstrated BALF levels of lactate dehydrogenase (LDH), beta-glucuronidase (BG), N-acetylglucosaminidase (NAG), and total protein (TP) increased in a dose-related fashion at the earlier time points for all test materials, with the magnitude of change being greatest for silica. The temporal response for these parameters was significantly different for the two classes of materials. With time, the response for the fibrogenic dust steadily increased, while the levels for the nonfibrogenic dusts decreased toward normal values during the 2-month study period. Of the cellular changes, total cell numbers, neutrophils, and lymphocyte numbers were the most sensitive markers of the pulmonary response. As shown with the biochemical parameters, the cellular response to silica increased with time while that of the nuisance dusts did not. It was also found that, similar to inhalation studies, high doses of a nuisance dust may result in toxicity/inflammation. This toxicity at high dose levels emphasizes the importance of choosing relevant doses when comparing potentially fibrogenic and nonfibrogenic dusts. In conclusion, the persistent and progressive changes seen in the biochemical (LDH, TP, BG, NAG) and cellular parameters (total cells, neutrophils and lymphocytes) following silica administration correlated with the fibrotic response which occurred after exposure to this material. The less dramatic and transient changes seen with aluminum oxide and titanium dioxide correlated with the inert nature of these nuisance dusts. The results of this study indicate evaluation of BALF may provide a means to predict the chronic pulmonary response to a material.

TNFα and increased chemokine expression in rat lung after particle exposure

Abstract Macrophage inflammatory protein 2 (MIP-2) and CINC (Cytokine-Induced-Neutrophil-Chemoattractant) are members of the chemokine family of inflammatory and immunoregulatory cytokines. MIP-2 and CINC exhibit potent neutrophil chemotactic activity and are thought to be key mediators of inflammatory cell recruitment in response to tissue injury and infection. In the present studies, we examined the potential involvement of MIP-2 and CINC in particle-elicited inflammation in the rat lung and the role of TNFα in particle-induced chemokine expression. Acute intratracheal instillation exposure of F344 rats to α quartz or titanium dioxide was shown to markedly increase steady-state levels of MIP-2 and CINC mRNA in lung tissue; a response which was associated with a significant increase in neutrophils in the bronchoalveolar lavage fluid. Additional studies demonstrated that acute inhalation of crocidolite fibers by rats also induced increased MIP-2 and CINC expression. Since previous studies had demonstrated that TNFα stimulates MIP-2 and CINC expression in vitro and that particle exposure induces TNFα production in rat lung we examined the role of TNFα in a quartz-induced MIP-2 gene expression. We demonstrated that passive immunization of mice against TNFα markedly attenuated the increased lung MIP-2 mRNA seen in response to a quartz inhalation. Collectively, these findings suggest that the chemokines MIP-2 and CINC play a role in neutrophil recruitment to the rat lung after particle exposure and indicate that particle-induced expression of these chemokines is mediated, at least in part, by production of TNFα.

Particles, inflammation and respiratory tract carcinogenesis

Particle-induced carcinogenesis is a non-specific outcome of many different particles. It was the purpose of this study, (i) to comprehensively review some of the mechanisms through which particles and particle-associated carcinogens can cause mutagenic/carcinogenic effects, and (ii) to indicate how this affects risk assessment studies. Data are presented that demonstrate the crucial role of a chronic inflammatory response in mutagenic effects of both silica and carbon black particles on the HPRT gene in lung target cells. The concept of inflammation in particle-induced genotoxicity is put into the context of other mechanisms, such as the release of cytokines and reactive oxygen species. It is concluded that interpretation of rat inhalation studies should certainly include this concept.

Respiratory tract responses to dust: Relationships between dust burden, lung injury, alveolar macrophage fibronectin release, and the development of pulmonary fibrosis

A multidisciplinary approach was used to investigate the responses of the respiratory tract to silica (SiO2) or titanium dioxide (TiO2). Rats were intratracheally instilled with 5-100 mg/kg of dust and bronchoalveolar lavage fluid (BALF) lactate dehydrogenase (LDH) and total protein (TP) and ex vivo alveolar macrophage (AM) fibronectin release assessed on Days 7, 14, and 28 after exposure. Lung dust burdens were determined on Days 1, 7, and 28 after instillation. Both dusts elicited dose-related increases in BALF LDH and TP, a response which was more pronounced and progressive with SiO2. All doses of SiO2 elicited persistent increases in AM fibronectin release. TiO2 stimulated persistent increases in AM fibronectin release at greater than or equal to 50 mg/kg, with transient or no effect at less than or equal to 10 mg/kg. Increased SiO2 retention was observed for all doses and TiO2 retention was increased only at doses greater than or equal to 50 mg/kg. In vitro exposure of naive AM to SiO2 or TiO2 did not stimulate AM fibronectin release. Histopathology demonstrated fibrosis at all SiO2 doses; only TiO2 doses greater than or equal to 50 mg/kg resulted in fibrosis. These results reveal an association between increased dust retention, lung injury, activation of AM fibronectin release, and the development of fibrosis. The magnitude and temporal pattern of responses clearly differentiated SiO2 from TiO2. The correlation of BALF markers of lung injury and increased AM fibronectin release with the development of fibrosis supports the use of these parameters as predictive biomarkers of dust-induced interstitial lung disease.

Chemokine regulation of ozone-induced neutrophil and monocyte inflammation

Pulmonary inflammation has been observed in humans and in many animal species after ozone exposure. Inflammatory cell accumulation involves local synthesis of chemokines, including neutrophil chemoattractants such as macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractants, such as monocyte chemoattractant protein-1 (MCP-1). To better understand the mechanism of ozone-induced inflammation, we exposed mice and rats to ozone for 3 h and measured MIP-2 and MCP-1 gene expression. In C57BL/6 mice, steady-state mRNA levels for MCP-1 in the lung increased at 0.6 parts/million (ppm) ozone and were maximal at 2.0 ppm ozone. After exposure to 2 ppm ozone, MIP-2 mRNA levels peaked at 4 h postexposure, whereas MCP-1 mRNA levels peaked at 24 h postexposure. Neutrophils and monocytes recovered in bronchoalveolar lavage fluid peaked at 24 and 72 h, respectively. The accumulation of monocytes was thus delayed relative to that of neutrophils, consistent with the sequential expression of the corresponding chemokines. The role of MCP-1 in monocyte accumulation was evaluated in greater detail in rats. Ozone caused an increase in monocyte chemotactic activity in bronchoalveolar fluid that was inhibited by an antibody directed against MCP-1. Ozone-induced MCP-1 mRNA levels were higher in lavage cells than in whole lung tissue, indicating that lavage cells are an important source of MCP-1. In these cells, nuclear factor-κB, a nuclear transcription factor implicated in MCP-1 gene regulation, was also activated 20-24 h after ozone exposure. These findings indicate that monocyte accumulation subsequent to acute lung injury can be mediated through MCP-1 and that nuclear factor-κB may play a role in ozone-induced MCP-1 gene expression.Pulmonary inflammation has been observed in humans and in many animal species after ozone exposure. Inflammatory cell accumulation involves local synthesis of chemokines, including neutrophil chemoattractants such as macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractants, such as monocyte chemoattractant protein-1 (MCP-1). To better understand the mechanism of ozone-induced inflammation, we exposed mice and rats to ozone for 3 h and measured MIP-2 and MCP-1 gene expression. In C57BL/6 mice, steady-state mRNA levels for MCP-1 in the lung increased at 0.6 parts/million (ppm) ozone and were maximal at 2.0 ppm ozone. After exposure to 2 ppm ozone, MIP-2 mRNA levels peaked at 4 h postexposure, whereas MCP-1 mRNA levels peaked at 24 h postexposure. Neutrophils and monocytes recovered in bronchoalveolar lavage fluid peaked at 24 and 72 h, respectively. The accumulation of monocytes was thus delayed relative to that of neutrophils, consistent with the sequential expression of the corresponding chemokines. The role of MCP-1 in monocyte accumulation was evaluated in greater detail in rats. Ozone caused an increase in monocyte chemotactic activity in bronchoalveolar fluid that was inhibited by an antibody directed against MCP-1. Ozone-induced MCP-1 mRNA levels were higher in lavage cells than in whole lung tissue, indicating that lavage cells are an important source of MCP-1. In these cells, nuclear factor-kappa B, a nuclear transcription factor implicated in MCP-1 gene regulation, was also activated 20-24 h after ozone exposure. These findings indicate that monocyte accumulation subsequent to acute lung injury can be mediated through MCP-1 and that nuclear factor-kappa B may play a role in ozone-induced MCP-1 gene expression.

Stimulation of rat alveolar macrophage fibronectin release in a cadmium chloride model of lung injury and fibrosis

Rats were exposed to saline or cadmium chloride (CdCl2) at 25, 100, or 400 micrograms/kg body weight by intratracheal instillation. At 3, 7, 14, and 28 days after exposure five animals/treatment were euthanized, the lungs were lavaged, and bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH), total protein, N-acetylglucosamindase (NAG), and cell number, type, and viability. Lung hydroxyproline concentration was characterized as a marker of lung collagen. Alveolar macrophages (AM) obtained in BALF were cultured and the release of fibronectin and TNF was determined. Lung tissue was examined microscopically at 28 and 90 days after exposure. Exposure to CdCl2 resulted in lung injury and inflammation demonstrated by increases in BALF LDH, total protein, NAG, and inflammatory cells. AM TNF release was not significantly changed by CdCl2 treatment. All doses of CdCl2 stimulated AM fibronectin secretion, a response which persisted throughout the 28-day postexposure period examined. Pulmonary fibrosis was demonstrated biochemically and/or histologically (trichrome staining tissue) at all CdCl2 dose levels. The association of CdCl2-induced AM fibronectin release with lung fibrosis confirms and extends previous observations relating AM-derived fibronectin to the development of interstitial lung disease and provides further evidence that the persistent increase in AM fibronectin release represents an early indicator of fibrosis.

ESTABLISHMENT OF IMMORTALIZED ALVEOLAR TYPE II EPITHELIAL CELL LINES FROM ADULT RATS

SummaryWe developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37° C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham’s F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number. However, at later passages the 6T cells became polyploid, while the 6TN genotype remained stable. The RLE-6T and 6TN cells were not tumorigenic in nude mice. The cell isolation methods reported and the novel cell lines produced represent potentially useful tools to study the role of pulmonary epithelial cells in neoplastic and nonneoplastic lung disease.