Protection from pulmonary fibrosis in the absence of CCR2 signaling.

Pulmonary fibrosis can be modeled in animals by intratracheal instillation of FITC, which results in acute lung injury, inflammation, and extracellular matrix deposition. We have previously shown that despite chronic inflammation, this model of pulmonary fibrosis is lymphocyte independent. The CC chemokine monocyte-chemoattractant protein-1 is induced following FITC deposition. Therefore, we have investigated the contribution of the main monocyte-chemoattractant protein-1 chemokine receptor, CCR2, to the fibrotic disease process. We demonstrate that CCR2(-/-) mice are protected from fibrosis in both the FITC and bleomycin pulmonary fibrosis models. The protection is specific for the absence of CCR2, as CCR5(-/-) mice are not protected. The protection is not explained by differences in acute lung injury, or the magnitude or composition of inflammatory cells. FITC-treated CCR2(-/-) mice display differential patterns of cellular activation as evidenced by the altered production of cytokines and growth factors following FITC inoculation compared with wild-type controls. CCR2(-/-) mice have increased levels of GM-CSF and reduced levels of TNF-alpha compared with FITC-treated CCR2(+/+) mice. Thus, CCR2 signaling promotes a profibrotic cytokine cascade following FITC administration.

Role of diminished epithelial GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis.

Evidence derived from human and animal studies strongly supports the notion that dysfunctional alveolar epithelial cells (AECs) play a central role in determining the progression of inflammatory injury to pulmonary fibrosis. We formed the hypothesis that impaired production of the regulatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) by injured AECs plays a role in the development of pulmonary fibrosis. To test this hypothesis, we used the well-characterized model of bleomycin-induced pulmonary fibrosis in rats. GM-CSF mRNA is expressed at a constant high level in the lungs of untreated or saline-challenged animals. In contrast, there is a consistent reduction in expression of GM-CSF mRNA in the lung during the first week after bleomycin injury. Bleomycin-treated rats given neutralizing rabbit anti-rat GM-CSF IgG develop increased fibrosis. Type II AECs isolated from rats after bleomycin injury demonstrate diminished expression of GM-CSF mRNA immediately after isolation and in response to stimulation in vitro with endotoxin compared with that in normal type II cells. These data demonstrate a defect in the ability of type II epithelial cells from bleomycin-treated rats to express GM-CSF mRNA and a protective role for GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis.

Deficient in vitro and in vivo phagocytosis of apoptotic T cells by resident murine alveolar macrophages.

Apoptotic lymphocytes are readily identified in murine lungs, both during the response to particulate Ag and in normal mice. Because apoptotic lymphocytes are seldom detected in other organs, we hypothesized that alveolar macrophages (AMphi) clear apoptotic lymphocytes poorly. To test this hypothesis, we compared in vitro phagocytosis of apoptotic thymocytes by resident AMphi and peritoneal macrophages (PMphi) from normal C57BL/6 mice. AMphi were deficient relative to PMphi both in percentage containing apoptotic thymocytes (19.1 +/- 1% vs 96 +/- 2.6% positive) and in phagocytic index (0.23 +/- 0.02 vs 4.2 +/- 0.67). This deficiency was not due to kinetic differences, was seen with six other inbred mouse strains, and was not observed using carboxylate-modified polystyrene microbeads. Annexin V blockade indicated that both Mphi types cleared apoptotic T cells by a mechanism involving phosphatidylserine expression. By contrast, neither mAb blockade of a variety of receptors (CD11b, CD29, CD51, and CD61) known to be involved in clearance of apoptotic cells, nor the tetrapeptide RGDS (arginine-glycine-aspartic acid-serine) blocked ingestion by either type of macrophage. To confirm these studies, apoptotic thymocytes were given intratracheally or i.p. to normal mice, and then AMphi or PMphi were recovered 30-240 min later. Ingestion of apoptotic thymocytes by AMphi in vivo was significantly decreased at all times. Defective ingestion of apoptotic lymphocytes may preserve AMphi capacity to produce proinflammatory cytokines in host defense, but could contribute to development of autoimmunity by failing to eliminate nucleosomes.

Weight gain after lung volume reduction surgery is not correlated with improvement in pulmonary mechanics.

STUDY OBJECTIVES: Malnutrition and low body weight are common in patients with emphysema. Previous work has demonstrated correlation between severity of airflow obstruction and body weight. Lung volume reduction surgery (LVRS) is a recent advance in the treatment of patients with severe emphysema that results in improved pulmonary function. We formed the hypothesis that improved lung mechanics after LVRS would result in body weight gain. DESIGN: Retrospective chart review. PATIENTS: All patients who underwent bilateral LVRS for severe emphysema at the University of Michigan between January 1995 and April 1996 were eligible for the study. MEASUREMENTS AND RESULTS: Pulmonary function and body weight were measured preoperatively and at 3, 6, and 12 months postoperatively for patients who underwent bilateral LVRS between January 1995 and April 1996. The average weight gain in 38 patients returning for 12 months of follow-up was 3.8 +/- 0.9 kg, or 6.2% of the preoperative weight. Women gained significantly more weight than men (9.2 vs 2.2%, respectively) at 1 year. Interestingly, there was no correlation between change in weight and postoperative change in FEV(1), FVC, residual volume (RV), total lung capacity (TLC), or RV/TLC at 12 months. However, there was a statistically significant correlation between weight gained and improvement in diffusion of carbon monoxide measured 12 months postoperatively. CONCLUSIONS: This study shows that patients with severe emphysema gain weight after LVRS. These changes were independent of changes in pulmonary mechanics but may be a result of improved gas exchange. These findings provide further information about benefits of LVRS in patients with advance emphysema that are beyond simple changes in pulmonary function.

Induction of lung fibrosis in the mouse by intratracheal instillation of fluorescein isothiocyanate is not T-cell-dependent.

Pulmonary fibrosis is the pathological result of a diverse group of insults. Common features of this group of diseases include chronic inflammation and immune cell activation. The pathogenesis of pulmonary fibrosis is not well defined and the prognosis is poor, highlighting the need for good animal models to elucidate the cellular and molecular events that lead to pulmonary fibrosis. This paper provides insight on a newly described model of pulmonary fibrosis using a single intratracheal challenge with fluorescein isothiocyanate (FITC). Balb-c and C57BL6 mice given intratracheal FITC develop acute lung injury followed by chronic inflammation. Significant increases in lung collagen content compared to saline-treated mice are noted at day 21 after inoculation. T-cell-deficient animals develop similar increases in lung collagen content compared to immunocompetent controls despite the abrogation of specific anti-FITC serum antibodies. Thus, the induction of fibrosis in FITC-challenged mice is not dependent on T cell immunity. Persistent chronic inflammation and acute lung injury may be the inciting events for the development of lung fibrosis in this model.

Induction of ICAM-1 expression on alveolar epithelial cells during lung development in rats and humans.

Intercellular adhesion molecule-1 (ICAM-1) is an adhesion protein involved in immune and inflammatory cell recruitment and activation. In normal, uninflamed adult rat lung, ICAM-1 is expressed at high levels on type I alveolar epithelial cells and is minimally expressed on type II cells. ICAM-1 expression by alveolar epithelial cells in vitro is a function of the state of cellular differentiation, and is regulated by factors influencing cell shape. Based upon this observation, we hypothesized that ICAM-1 expression by fetal lung epithelial cells is developmentally regulated. To investigate this hypothesis, rat and human lung tissues were obtained at time points that represent the canalicular, saccular, and alveolar stages of development. The relative expression of ICAM-1 protein and mRNA were determined in rat lungs from gestational days 18 and 21 (term = 22 days), from day 8 neonatal rats, and from adult rats. ICAM-1 protein was detectable at low level on day 18 and increased progressively during development. Relative expression of ICAM-1 protein was maximal in adult lung. Expression of ICAM-1 mRNA paralleled that of ICAM-1 protein. By immunohistochemical methods in rat and human lung, ICAM-1 was expressed at low level on cuboidal and flattening epithelial cells in the developing alveolar space at the canalicular and saccular stages; however, ICAM-1 expression was increased as epithelial cells spread and flattened during alveolarization. ICAM-1 was predominantly expressed on type I cells rather than type II cells at the alveolar stage in both the rat and human lungs. Thus, relative ICAM-1 expression progressively increased during lung development. ICAM-1 expression is correlated with the increase in surface area as alveolar structures develop and type I cell differentiation takes place. These data indicate that alveolar epithelial cell ICAM-1 expression is developmentally regulated.

Preoperative echocardiographic evaluation of patients referred for lung volume reduction surgery.

BACKGROUND: The most efficient preoperative assessment for lung volume reduction surgery (LVRS) in patients with advanced emphysema is undefined. This study analyzed the preoperative assessment of patients by surface echocardiography (without and with dobutamine infusion), the results of which were used to exclude patients with significant pre-existing cardiac disease, a contraindication to LVRS, from the surgery. SETTING: A university-based, tertiary care referral center. METHODS: Patients with emphysema who met initial LVRS screening criteria underwent resting and stress surface echocardiography with Doppler imaging. Patients were evaluated prospectively for perioperative cardiac complications. RESULTS: Between July 1994 and December 1996, 503 candidates for LVRS were evaluated. Of these, 207 patients (81.8%) who had echocardiography performed at our institution formed the primary study group. Images were adequate for the analysis of chamber sizes and function in 206 patients (99.5%) undergoing resting echocardiography, and the images were adequate for wall motion analysis in 172 of 174 patients (98.9%) undergoing functional testing. Right heart abnormalities were common (40.1%). Significant pulmonary hypertension (> 35 mm Hg) was uncommon (5 patients, 5.4%) among the 92 patients who subsequently underwent right heart catheterization. Occult ischemia, left ventricular dysfunction, and valvular abnormalities also were uncommon. Thus, although Doppler imaging estimates of right ventricular systolic pressure were imperfect, echocardiographic findings of normal right heart anatomy and function excluded significant pulmonary hypertension. Ninety patients (43%) eventually underwent LVRS (70 bilateral and 20 unilateral). A total of 13 perioperative cardiac events occurred in 10 patients, 6 of whom had undergone preoperative echocardiography. No patient suffered acute myocardial infarction or cardiac death. CONCLUSIONS: Despite potential limitations due to severe obstructive lung disease, surface echocardiographic imaging is a feasible, noninvasive tool in this patient population to identify patients with evidence of cor pulmonale that suggests pulmonary hypertension. The routine use of surface resting and stress echocardiography for preoperative screening obviates the need for invasive right heart catheterization in many patients and results in a low incidence of significant perioperative cardiac complications.

Chemotaxis of alveolar macrophages in response to signals derived from alveolar epithelial cells.

We have postulated that alveolar epithelial cells (AEC) play a critical role in local regulation of alveolar macrophage (AM) recruitment and activation for host defense in the lung. The present study explores the effects of conditioned medium from AEC (AEC-CM) on the migration of AM, using a Boyden chamber assay. AEC-CM was chemotactic for AM, with peak activity observed with a 1:10 dilution. We previously showed that rat AEC express the chemokines RANTES (regulated on activation, normal T expressed and secreted) and monocyte chemoattractant protein 1 (MCP-1) as well as granulocyte-macrophage colony-stimulating factor (GM-CSF). Neutralizing antibodies to RANTES and to MCP-1 and immunoprecipitation of GM-CSF decreased the chemotactic activity of AEC-CM by 58%, 29%, and 47%, respectively. Similar levels of chemotaxis were found in response to recombinant RANTES, MCP-1, and GM-CSF. In each instance the optimal dose was very low (0.01 to 0.1 ng/ml), with diminished chemotaxis at higher doses. Peritoneal macrophages (PM) also migrated in response to AEC-CM and each of the recombinant cytokines; however, AM were much more sensitive to AEC-CM, RANTES, and GM-CSF than were PM. AM migrated preferentially from medium conditioned by unstimulated AEC toward supernatants from interleukin 1alpha-stimulated AEC. Therefore, AEC may control the distribution of AM through the creation of local chemotactic gradients and are likely to play a critical role in the host response to low-level antigen entry into the peripheral lung.

Lung volume reduction surgery alters management of pulmonary nodules in patients with severe COPD.

OBJECTIVE: To examine the role of lung volume reduction surgery (LVRS) in expanding the treatment options for patients with single pulmonary nodules and emphysema. METHODS: Retrospective review of all patients undergoing LVRS at the University of Michigan between January 1995 and June 1996. Those undergoing simultaneous LVRS and resection of a suspected pulmonary malignancy formed the study group and underwent history and physical examination, pulmonary function tests, chest radiography, and high-resolution CT of the chest. If heterogeneous emphysema was found, cardiac imaging and single-photon emission CT perfusion lung scanning were performed. All study patients participated in pulmonary rehabilitation preoperatively. Age- and sex-matched patients who had undergone standard lobectomy for removal of pulmonary malignancy during the same period formed the control group. RESULTS: Of 75 patients who underwent LVRS, 11 had simultaneous resection of a pulmonary nodule. In 10 patients, the nodules were radiographically apparent with 1 demonstrating central calcification. Histologic evaluation revealed six granulomas, two hamartomas, and three neoplastic lesions (one adenocarcinoma, one squamous cell, and one large cell carcinoma). Preoperative FEV1 was 26.18+/-2.49% predicted in the LVRS group and 81.36+/-6.07% predicted (p=0.000001) in the control group, and the FVC was 65.27+/-5.17% predicted vs 92.18+/-5.53% predicted (p=0.002). Two LVRS patients had a PaCO2 >45 mm Hg while 11 exhibited oxygen desaturation during a 6-min walk test. Postoperative complications occurred in two LVRS patients and three control patients. The mean length of stay in the LVRS group (7.55+/-1.10 days) was not different than in the control group (8.81+/-1.56 days). Three months after LVRS and simultaneous nodule resection, FEV1 rose by 47%, FVC by 25%, and all study patients noted less dyspnea as measured by transitional dyspnea index. CONCLUSIONS: Simultaneous LVRS and resection of a suspected bronchogenic carcinoma is feasible and associated with minimal morbidity and significantly improved pulmonary function and dyspnea.

Association of ICAM-1 with the cytoskeleton in rat alveolar epithelial cells in primary culture.

Intercellular adhesion molecule-1 ICAM-1) is a transmembrane adhesion protein that is expressed constitutively on the apical surface of type I cells in vivo and on type II cells in vitro as they spread in culture, assuming type I cell-like characteristics. To investigate the possible interaction of ICAM-1 with the alveolar epithelial cell cytoskeleton, rat type II cells in primary culture were extracted with nonionic detergent, and residual ICAM-1 associated with the cytoskeletal remnants was determined using immunofluorescence microscopy, immunoprecipitation, and cell-based enzyme-linked immunosorbent assay. A large fraction of alveolar epithelial cell ICAM-1 remained associated with the cytoskeleton after detergent extraction, whereas two other transmembrane molecules, transferrin receptor and class II major histocompatibility complex, were completely removed. ICAM-1 was redistributed on the cell surface after the disruption of actin filaments with cytochalasin B, suggesting interaction with the actin cytoskeleton. In contrast, ICAM-1 was completely detergent soluble in rat pulmonary artery endothelial cells, human umbilical vein endothelial cells, and rat alveolar macrophages. The association of ICAM-1 with the alveolar epithelial cell cytoskeleton was not altered after stimulation with inflammatory cytokines. However, detergent resistant ICAM-1 was significantly increased after crosslinking of ICAM-1 on the cell surface, suggesting that this cytoskeletal association may be modulated by interactions of alveolar epithelial cells with inflammatory cells. The association of ICAM-1 with the cytoskeleton in alveolar epithelial cells may provide a fixed intermediary between mobile inflammatory cells and the alveolar surface.

Regulation of rat pulmonary dendritic cell immunostimulatory activity by alveolar epithelial cell-derived granulocyte macrophage colony-stimulating factor.

The presentation and recognition of foreign antigen is the critical initial event in the development of local immunity. In the lung, antigen-presenting cell activity is largely attributable to pulmonary dendritic cells (DC) that are distributed along the airways and throughout the pulmonary interstitium in close proximity to overlying alveolar epithelial cells. To test the hypothesis that DC immunostimulatory activity might be locally regulated by overlying alveolar epithelial cells, we evaluated the ability of rat type II alveolar epithelial cells to influence the capacity of purified rat pulmonary DC to stimulate T-cell proliferation in an allogeneic, mixed leukocyte reaction. We found that alveolar epithelial cells greatly enhanced the ability of dendritic cells to induce T-cell proliferation. This effect on DC immunostimulatory activity was mediated by a soluble factor preferentially secreted from the basolateral epithelial cell surface. Alveolar epithelial cultures were found to express mRNA for granulocyte macrophage colony-stimulating factor (GM-CSF), and blocking antibodies against GM-CSF partially neutralized the effect of epithelial cell-conditioned media on DC stimulatory activity, indicating that the effect was due at least in part to alveolar epithelial cell-derived GM-CSF. Through the polar secretion of GM-CSF, alveolar epithelial cells may play an important role in creating distinct immunologic environments within the lung.

Disparate cytokine regulation of ICAM-1 in rat alveolar epithelial cells and pulmonary endothelial cells in vitro.

Intercellular adhesion molecule-1 (ICAM-1) is expressed at high levels on type I alveolar epithelial cells in the normal lung and is induced in vitro as type II cells spread in primary culture. In contrast, in most nonhematopoetic cells ICAM-1 expression is induced in response to inflammatory cytokines. We have formed the hypothesis that the signals that control ICAM-1 expression in alveolar epithelial cells are fundamentally different from those controlling expression in most other cells. To test this hypothesis, we have investigated the influence of inflammatory cytokines on ICAM-1 expression in isolated type II cells that have spread in culture and compared this response to that of rat pulmonary artery endothelial cells (RPAEC). ICAM-1 protein, determined both by a cell-based enzyme-linked immunosorbent assay and by Western blot analysis, and mRNA were minimally expressed in unstimulated RPAEC but were significantly induced in a time- and dose-dependent manner by treatment with tumor necrosis factor-alpha, interleukin-1 beta, or interferon-gamma. In contrast, these cytokines did not influence the constitutive high level ICAM-1 protein expression in alveolar epithelial cells and only minimally affected steady-state mRNA levels. ICAM-1 mRNA half-life, measured in the presence of actinomycin D, was relatively long at 7 h in alveolar epithelial cells and 4 h in RPAEC. The striking lack of response of ICAM-1 expression by alveolar epithelial cells to inflammatory cytokines is in contrast to virtually all other epithelial cells studied to date and supports the hypothesis that ICAM-1 expression by these cells is a function of cellular differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Lung sources and cytokine requirements for in vivo expression of inducible nitric oxide synthase.

Products of inducible nitric oxide synthase (iNOS) are known to be involved in lung injury following intrapulmonary deposition of immunoglobulin G immune complexes (IgG-ICx). In the current studies rat alveolar macrophages stimulated in vitro with murine interferon gamma (IFN-gamma), tumor necrosis factor alpha, interleukin 1 alpha, (IL-1 alpha), lipopolysaccharide (LPS), or IgG-ICx immunostained for iNOS and produced nitrite/nitrate- (NO2-/NO3-) in a dose- and time-dependent manner requiring availability of L-arginine. Under the same conditions, IL-4 and IL-10 reduced NO2-/NO3- generation. Type II alveolar epithelial cells, which were obtained from normal rat lungs and stimulated in vitro with IgG-ICx, LPS, or IFN-gamma, also immunostained for iNOS and generated NO2-/NO3-. Special techniques of bronchoalveolar lavage (BAL) were used to retrieve alveolar macrophages and type II alveolar epithelial cells. Under these conditions, intrapulmonary deposition of LPS yielded BAL fluids containing increased amounts of NO2-/NO3- and macrophages that spontaneously released NO2-/NO3- and stained for iNOS. After intrapulmonary deposition of IgG both macrophages as well as type II cells (retrieved by BAL) spontaneously produced NO2-/NO3- and both cell types immunostained for iNOS (approximately 20% of all type II cells and 35% of all alveolar macrophages). Using dual fluorescence staining for cell identification, frozen sections of lung tissue after IgG immune complex deposition revealed iNOS in both alveolar macrophages and type II cells. Finally, in the immune complex model of alveolitis, the appearance of iNOS in macrophages as well as macrophage production in vitro of NO2-/NO3- was dependent on the in vivo availability of tumor necrosis factor alpha, IL-1, and IFN-gamma. These studies suggest a dual cell source for nitric oxide in inflamed lungs and the requirements for iNOS of several cytokines.

Cloning the rat homolog of the CD28/CTLA-4-ligand B7-1: structural and functional analysis.

T cell activation involves the delivery of two independent signals to the naive T cell. The first signal occurs with engagement of the TCR. One of the best characterized second signals is ligation of CD28 on the surface of T cells by B7 molecules (B7-1, B7-2) present on the surface of activated antigen presenting cells (APCs). Recent studies have demonstrated that injection of a human fusion protein, CTLA-4-Ig, which in humans binds to both B7-1 and B7-2, prevents cardiac allograft rejection in a rat transplantation model when given 48 h after engraftment. In order to better characterize the role of B7-1 (which is maximally expressed 48 h after activation of APCs) in this model, as well as in models of tumor-induced immune responses, we have cloned the rat homolog of B7-1, and now report on its structure and function. A 1030 bp cDNA containing the entire coding sequence of the rat B7-1 was cloned with a polymerase chain reaction strategy utilizing degenerate primers derived from published murine and human B7-1 sequences. The rat B7-1 coding sequence is 67 and 81% homologous to human and murine B7-1 cDNAs, and the predicted peptide sequence is likewise 57 and 66% identical to the peptide sequences of human and murine B7-1 respectively. The greatest area of identity occurs in the extracellular portion of the molecule, particularly the Ig-C like domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiopulmonary exercise testing following allogeneic lung transplantation for different underlying disease states.

OBJECTIVES: To assess the exercise response to single lung transplantation in chronic airflow obstruction (CAO), idiopathic pulmonary fibrosis (IPF), and pulmonary vascular disease (PVD) vs double lung transplantation at well-defined time points after transplantation, and to define the change in exercise response in SLT and DLT over the first year after transplantation. DESIGN: Prospective study. SETTING: Tertiary referral hospital. PATIENTS: Fourteen stable SLT recipients (6 with CAO, 4 with IPF, 4 with PVD) and 11 stable DLT recipients. MEASUREMENTS: Spirometry, lung volumes, diffusion lung capacity for carbon monoxide (DLco) and MVV measured prior to exercise at 3 months (n = 25) then at 3-month intervals up to a maximum of 12 months post-transplantation (n = 18 [12 SLT and 6 DLT]). Symptom-limited cardiopulmonary exercise tests at same time points (n = 25 at 3 months, n = 18 [12 SLT and 6 DLT] at 3-month intervals up to 12 months). Breathlessness was estimated by visual analogue scale prior to exercise and at peak exercise. RESULTS: At 3 months, FEV1 percent predicted was lower for SLT-CAO and SLT-IPF vs DLT (p < or = 0.05). Mean FEV1/FVC was lower for SLT-CAO vs all other groups (p < or = 0.05). The FVC, MVV, and DLco/VA were similar for all groups. The TLC and RV were higher for the SLT-CAO group compared with all others. The TLC was lower for SLT-PVD compared with DLT. Exercise responses were similar in all groups studied without a statistically significant difference in achieved VO2, work rate, O2 pulse, anaerobic threshold, heart rate response, respiratory rate, VE/MVV, and VT/VC. The change in O2 saturation during exercise was the least in recipients of DLT. Maximal achieved VO2 rose from 3 to 6 months after SLT but dropped by 9 to 12 months after transplantation. Maximal achieved VO2 trended up from 3 to 6 months after DLT but dropped by 9 to 12 months after transplantation. Maximal achieved work rate rose in both SLT and DLT from 3 to 9 to 12 months after transplantation. There was no significant difference in breathlessness at rest and peak exercise measured between recipients of SLT or DLT. CONCLUSIONS: Minor differences in pulmonary function and change in O2 saturation occur between recipients of SLT and DLT during the first posttransplant year. These differences are most pronounced when comparing SLT-CAO with DLT. However, there is no significant difference in exercise capacity between SLT for CAO, IPF, PVD, and DLT. The rise in maximum achieved VO2 over the first 6 months after transplantation may reflect the effects of exercise training and should be taken into account when examining aerobic response after transplantation.

Regulation of the immunostimulatory activity of rat pulmonary interstitial dendritic cells by cell-cell interactions and cytokines.

Pulmonary dendritic cells (DC) are potent antigen-presenting cells that are thought to play a critical role in the initiation of immune responses within the lung. Because the lung is both a site of entry into the body for microbial pathogens and the organ of gas exchange, pulmonary immune responses must be meticulously regulated to achieve a balance between host defense and respiration. The initial interaction of DC with T cells in the lung is an excellent point at which to control local immune responses. Studies of the regulation of DC accessory cell function have been greatly hampered by difficulties in obtaining pure populations of pulmonary DC that have not been subjected to prolonged incubations during which the DC may undergo functional alteration. We now describe a method for isolating pulmonary DC from the rat that yields 1 x 10(5) cells/rat with > 90% purity. These cells are potent accessory cells, inducing T cell proliferation in a mixed leukocyte reaction (MLR) at a stimulator-to-responder ratio of 1:1,000. This method, which involves flow cytometric separation of nonphagocytic cells that stain brightly for class II MHC (OX6) from a population of low-density pulmonary interstitial cells, avoids extended incubations at 37 degrees C and thus allows study of a relatively pure population of cells that have functional capacities resembling those of naive cells from the normal lung. With these cells, we demonstrate that the functional capacity of pulmonary DC as stimulator cells in an MLR is significantly increased by exposure to the cytokines interleukin-1 or granulocyte/macrophage colony-stimulating factor (GM-CSF) and by culture with interstitial, but not alveolar, macrophages. Furthermore, DC are heterogeneous with respect to the cell surface expression of receptor for GM-CSF, and this expression is subject to modulation in cell culture. From these studies, we conclude that the immunostimulatory capacity of pulmonary DC is a function of local interactions with cytokines and other parenchymal cells. This suggests that DC function may be an important regulatory point for the local control of pulmonary immune responses.

Effect of granulocyte-macrophage colony-stimulating factor on rat alveolar macrophage anticryptococcal activity in vitro.

Cryptococcus neoformans, a pathogenic fungus usually acquired by inhalation, causes the most common lethal mycosis in AIDS. The resident lung phagocytes, alveolar macrophages (AM phi), inhibit growth of C. neoformans poorly unless activated by cytokines such as IFN-gamma. In this study, we examined the effect of rat AM phi of the potent hematopoietic and M phi-activating cytokine, granulocyte-macrophage CSF (GM-CSF), alone and in combination with other cytokines. Rat AM phi monolayers were preincubated with 0.1 to 1000 U/ml GM-CSF without or with other recombinant cytokines, and then were incubated with viable C. neoformans (strain H99/C3D). Growth inhibition was assessed by counting cryptococcal CFU at 24 and 48 h of coculture; AM phi proliferation was assessed by measuring both uptake of [3H]TdR and AM phi numbers. AM phi preincubated with GM-CSF for 5 days (but not for shorter periods) inhibited growth of C. neoformans. Anticryptococcal activity required direct contact of AM phi with C. neoformans, but once induced by preincubation, did not require continued exposure to GM-CSF. Induction of anticryptococcal activity by GM-CSF was dose dependent (maximal induction at 250 U/ml), and was due to both increased ingestion and killing. GM-CSF induced AM phi proliferation, but anticryptococcal activity was not due totally to increases in AM phi numbers, indicating AM phi activation by GM-CSF. GM-CSF-induced AM phi proliferation was increased by IL-6, unchanged by IL-8, and abolished by LPS or IFN-gamma. However, IL-6 did not increase GM-CSF-induced anticryptococal activity. The combination of GM-CSF and IFN-gamma showed rapid and sustained anticryptococcal activity, unlike either cytokine alone. Our in vitro data suggest that the combination of GM-CSF and IFN-gamma may have beneficial effects on host defense against C. neoformans in vivo.

Septic pulmonary embolism due to periodontal disease.

Three weeks following a toothache, a 56-year-old man developed cough, sputum, fever, and pleuritic chest pain. He had mild periodontal disease and his chest radiographs and chest computed tomographic (CT) scans showed multiple pulmonary nodules. The CT scan strongly suggested septic pulmonary embolism. Aspirated pus from one of the nodules yielded pure growth of Streptococcus intermedius. Lesions resolved with antimicrobial therapy. The usual predisposing factors for septic pulmonary embolism were absent, and, the isolation of S intermedius from the pus, the antecedent toothache, and periodontal disease all suggested the gingiva as the source. We hypothesize that periodontal infection led to bacteremia, seeding of the lungs, and multiple anaerobic pulmonary abscesses, akin to reported instances of infective endocarditis from dental foci without any prior dental procedures. To our knowledge, this presentation of septic pulmonary embolism is unprecedented.

Characterization of the production of monocyte chemoattractant protein-1 and IL-8 in an allogeneic immune response.

Delayed-type hypersensitivity and allograft rejection are dependent upon the generation of Ag-specific T cell immune responses. The mixed lymphocyte reaction is a model of alloantigen-driven immunity and has provided significant insight into the mechanisms of T cell proliferation. Recently, soluble mediators, including TNF-alpha have been shown to be involved in the full expression of this response. In order to elucidate the potential mechanisms of cellular recruitment in the context of either delayed-type hypersensitivity or allograft rejection, we employed a mixed lymphocyte reaction to study the production of chemokines, monocyte chemoattractant protein-1 (MCP-1), and IL-8. Time-course experiments demonstrated that significant levels of MCP-1 and IL-8 were produced in allogeneic cultures as early as day 1, and that this relationship persisted over 6 days in culture. Northern blot analysis of chemokine mRNA confirmed the early induction of these genes in the allogeneic response. The levels of MCP-1 and IL-8 protein from mixed lymphocyte reaction supernatants as measured by specific ELISA were positively correlated with the proliferative response as measured by [3H]TdR uptake. Although significant MCP-1 and IL-8 were produced during a mixed lymphocyte reaction, these molecules did not participate in the proliferative response, as neutralizing antibodies to either MCP-1 or IL-8 had no effect on proliferation. In order to ascertain the mechanism for the induction of MCP-1 and IL-8 during the mixed lymphocyte reaction, experiments were performed in the presence of neutralizing anti-human TNF antibodies. Neutralization of TNF resulted in significant abrogation of MCP-1 and IL-8 production (75 +/- 5 and 87 +/- 2 percent, respectively). Cells isolated from the mixed lymphocyte reaction at day 6, demonstrated that MCP-1 and IL-8 Ag were localized to mononuclear phagocytes. These results demonstrate that potent chemokines, MCP-1 and IL-8, are produced during the evolution of a mixed lymphocyte reaction, and their induction is TNF-dependent.