Annette Badgett

Interstitial pulmonary macrophages produce platelet-derived growth factor that stimulates rat lung fibroblast proliferation in vitro.

Alveolar macrophages from humans and several animal species produce factors in vitro that modulate fibroblast growth and have been proposed as mediators of interstitial pulmonary fibrosis. Pulmonary interstitial macrophages (IMs) have not been studied previously in this regard. Pulmonary IMs were isolated from prelavaged rat lungs by enzymatic digestion of tissue and subsequent differential adherence of cells to culture dishes. The ability of IMs to release modulators of fibroblast growth into the culture medium was assessed by measuring [3H]thymidine incorporation into DNA and/or nuclear labeling of early‐passage rat lung fibroblasts exposed to medium conditioned by IMs. The percentages of nuclei labeled in fibroblast cultures exposed to interstitial macrophage–conditioned medium (IMCM) alone did not significantly differ from that observed in controls, but fibroblasts exposed to IMCM supplemented with 2% platelet‐poor plasma showed a 2.6‐fold increase in labeling, indicating that IMCM contains predominantly “competence” growth factor activity. Similar results were obtained using purified human platelet‐derived growth factor (PDGF). The level of growth factor activity released by IMs increased in cells that had phagocytized iron spheres during the culture period. In addition, fractionation of IMCM by high‐performance liquid chromatography demonstrated most of the growth factor activity at a relative molecular mass of about 35 kd. Subsequent quantitative analysis of the fractions by an enzyme immunoassay for PDGF demonstrated that IMCM contains a homologue of human PDGF. These results show that IMs are capable of producing a PDGF‐like growth factor for autologous fibroblasts and that release of this factor is enhanced by exposure to an insoluble inorganic particle. Because PDGF is a potent growth factor for fibroblasts and is released by IMs, it is essential to ask in future studies whether this or similar macrophage products play a significant role in mediating fibroblast proliferation in vivo.

Eosinophilic Lung Inflammation in Particulate-Induced Lung Injury. Technical Consideration in Isolating RNA for Gene Expression Studies

Particulate and other pollutant exposures are associated with lung injury and inflammation. The purpose of this study was to develop an approach by which intact RNA could be obtained from inflamed lung tissue from particulate-exposed animals in order to correlate injury with specific gene expression. Male Sprague Dawley (SD) and Fischer-344 (F-344) rats were intratracheally instilled with saline or residual oil fly ash (ROFA) particles, 8.3 mg/kg body weight in saline. At various time points following ROFA instillation, lungs were either lavaged or used for RNA isolation. ROFA exposure produced an increase in bronchoalveolar lavage fluid (BALF) neutrophils in both SD and F-344 rats. A time-dependent increase in eosinophils occurred only in SD rats but not in F-344 rats. Extraction of inflamed pulmonary tissue having a high influx of eosinophils for RNA using the conventional acid guanidinium thiocyanate phenol-chloroform (AGPC) procedure failed to provide undegraded RNA suitable for RT-PCR and Northern blot analysis of beta-actin mRNA expression. Mixing intact total RNA from saline control rat lungs with degraded RNA samples from inflamed lung yielded a gel profile of degraded RNA, indicating the presence of ribonuclease-like activity in the RNA extracted from lung tissues having eosinophil influx. Evidently, the conventional AGPC procedure failed to completely remove ribonuclease activity associated with ROFA-induced pulmonary eosinophil influx. This study reports a single-step modification to the AGPC extraction method that does not require additional reagents or additional precipitation steps for extracting undegraded RNA from nuclease-rich inflamed lung tissue. The aqueous layer resulting from mixing homogenate and chloroform is extracted a second time using an equal volume of AGPC buffer followed by addition of chloroform and centrifugation. The second aqueous phase is then treated as described in the conventional RNA extraction protocol. This simple and convenient modification does not require multiple precipitations of RNA and yields undegraded RNA from inflamed lung tissue with a slightly higher A260/A280 ratio without affecting overall RNA recovery. The results indicate that undegraded RNA could not be isolated using the routine AGPC-based isolation technique from lung tissue containing eosinophils following ROFA exposure. The degraded RNA preparations were unsuitable for gene expression studies. However, undegraded RNA can be isolated from these tissues by modifying the original AGPC RNA extraction procedure, which is suitable for gene expression analysis using northern blot and RT-PCR techniques.

Interferon-γ modulates lung macrophage production of PDGF-BB and fibroblast growth

Abstract Platelet-derived growth factor (PDGF) is a potent mediator of fibroblast proliferation and chemotaxis. We have studied here the cytokine interferon-γ (IFN-γ) which is known to prime macrophages for increased PDGF production. Thus, we postulated that IFN-γ would act as a positive regulator of PDGF-BB secretion by rat alveolar macrophages, and in addition we asked whether or not the IFN-γ (a known anti-mitogenic cytokine) would block the growth response of primary lung fibroblasts to the PDGF-BB. Macrophages incubated with IFN-γ or iron spheres alone for 24 h secreted 2.5-fold more PDGF-BB than control macrophages incubated in serum-free medium. Preincubation of macrophages with IFN-γ prior to the addition of iron spheres synergistically increased PDGF-BB production 2–10-fold after 24 h. In contrast, when IFN-γ was added to quiescent rat lung fibroblasts (RLFs) in the presence of PDGF-BB, the cytokine induced a concentration-dependent decrease in cell growth, while IFN-γ alone did not affect proliferation. [ 125 I]PDGF-BB receptor assays showed that neither preincubation nor coincubation of RLF with IFN-γ affected PDGF-BB binding to its receptors.