Asthma prevalence, cost, and adherence with expert guidelines on the utilization of health care services and costs in a state Medicaid population.

OBJECTIVE: To provide a descriptive analysis of asthma prevalence and costs in a Medicaid population and gauge the degree of adherence with expert guidelines for asthma medication management from the National Asthma Education and Prevention Program. DATA SOURCES: Kentucky Medicaid administrative data for 1996. STUDY DESIGN: A cross-sectional retrospective analysis was used to determine adherence with asthma medication guidelines and utilization of asthma-related health care services and costs. Multivariate logistic regression was used to determine the relationship between nonadherence with the guidelines and utilization of health care services. PRINCIPAL FINDINGS: Of the 530,000 Medicaid recipients, 24,365 (4.6 percent) were identified as having asthma. Average annual asthma-related costs ($616) accounted for less than 20 percent of total health care costs ($3,645). Nonadherence to the guidelines was prevalent. Less than 40 percent of the patients received a prescription for a rescue medication, and fewer than 10 percent of the patients who received daily inhaled short-acting beta-2 agonists were regular users of inhaled steroids. Nonadherence to the guidelines was associated with an increased risk of an asthma-related hospitalization (odds ratio = 1.5, p < .05). CONCLUSIONS: Guideline nonadherence was widespread and associated with an increase in exacerbations of asthma that resulted in hospitalizations. Asthma prevalence and utilization of health care services in a Medicaid population were similar to previous estimates reported nationally and in health maintenance organizations.

Murine dendritic cells infected with adenovirus vectors show signs of activation.

Dendritic cells (DC) are highly efficient antigen presenting cells being actively evaluated as vaccine components. A number of studies have shown adenovirus-mediated gene transfer to cultured DCs is feasible and that Ad-modified DCs are effective at inducing T cell immunity in vitro and establishing antitumor immunity in experimental tumor models in vivo. The current study evaluates the biologic effects of Ad infection on murine bone marrow-derived DCs (BMDC) in primary culture. Ad infection (MOI 200) of BMDC induced significant increases in IL 12 p40 protein in culture supernatants (6 x that of uninfected BMDC and similar to that observed with addition of LPS and CD40 crosslinking antibody). Supernatants from Ad infected BMDCs induced appreciable increases in IFNgamma from naive splenocytes in culture. Consistent with DC activation, FACs analysis showed BMDC infected with Ad vectors up-regulated the surface expression of B7-2, ICAM-1 and MHC II. Additional experiments evaluated the role of virus attachment, internalization and gene expression using IL-12 p40 production as a marker of DC activation. Neither heat-inactivated Ad nor peptides containing the RGD sequence (the primary component of Ad penton base which interacts with cell surface integrins) induced significant amounts of IL12 p40. In contrast, psoralen/UV-inactivated Ad showed similar levels of IL12 p40 production compared with intact Ad. These data suggest this phenomenon is dependent on viral entry into the cell and/or translocation to the nucleus, and is independent of either viral gene or transgene expression.

Beryllium-stimulated in vitro migration of peripheral blood lymphocytes.

Inhalation of beryllium (Be) induces both inflammatory and metal antigen-specific immune responses in the lungs characterized by mononuclear cell infiltration and granuloma formation (chronic beryllium disease, CBD). We tested the hypothesis that Be-salts might increase the in vitro migration of peripheral blood mononuclear cells (PBMC). PBMC are mixed cells, consisting of lymphocytes and monocytes. We compared their responses to populations of both purified blood lymphocytes, and purified blood monocytes. Purified blood monocytes and lymphocytes, isolated by Percoll gradients and centrifugal elutriation from normal human subjects (n = 6), were exposed to graded concentrations (0.01 to 100 microM) of BeSO4 or to the control metal-salt Al2(SO4)3. Migratory responses of stimulated PBMC were measured in Boyden Chambers. As controls, PBMC mixed cells or purified lymphocytes or purified monocytes were unstimulated or stimulated with a positive chemoattractant, Zymosan-A treated pooled, normal human serum (ZAS). The migration index (MI) was defined as the distance (micrometers) that cells migrated through a 5 micron filter. The MI for unstimulated PBMC mixed cells was 75+/-4 whereas the MI for ZAS-stimulated PBMC mixed cells was 124+/-4 (P < or = 0.05, Tukey-Kramer). The MI for BeSO4 -stimulated (100 microM) PBMC mixed cells was 136+/-4. The observed increase in the BeSO4-stimulated PBMC mixed cell migration was significant down to 0.1 microM BeSO4. BeSO4, BeCl2 and BeF2, tested at 100 and 10 microM, were equally effective at inducing PBMC mixed cell migration. Equimolar concentrations of Al2(SO4)3 were not as effective at inducing PBMC mixed cell migration, MI < 100 at 100 microM, and did not induce PBMC mixed cell migration at concentrations below 1 microM. The migration of purified monocytes through filters was not increased in response to either BeSO4 or Al2(SO4)3 compared to controls, but did respond to ZAS (MI = 100+/-4). Purified lymphocytes migrated in response to stimulation with all concentrations of BeSO4 tested (100 microM MI = 133+/-9), and Al2(SO4)3 (100 microM MI = 85+/-8). There were no significant differences in the MI for PBMC mixed cells or for purified lymphocytes at the concentrations of BeSO4 tested. Our data show that Be directly induces the in vitro migration of PBMC mixed cells and purified blood lymphocytes, and not purified blood monocytes, across a broad range of Be concentrations. This induction of migration was independent of the molecular form of the Be-salt. Inhaled Be, by promoting lymphocyte emigration to the lung, may create a microenvironment that favors a Be-antigen-specific T-lymphocyte response, chronic inflammation, and CBD.

Staphylococcal toxic shock syndrome toxin-1 inhibits monocyte apoptosis.

BACKGROUND: Chronic atopic dermatitis (AD) lesions are associated with colonization by exotoxin-producing Staphylococcus aureus. Evidence suggests that cytokine production in AD, particularly of GM-CSF, prolongs survival of both peripheral blood monocytes and dermal monocyte-macrophages, the predominate inflammatory cell in lesions caused by chronic AD. OBJECTIVE: We sought to determine whether the staphylococcal exotoxin, toxic shock syndrome toxin-1 (TSST-1), could stimulate prosurvival cytokine production in monocytes and thereby inhibit apoptosis. METHODS: Cultures of peripheral blood monocytes from normal donors and subjects with AD were incubated with various concentrations of TSST-1, and the incidence of apoptosis was assessed by examining cytospin preparations and the appearance of hypodiploid DNA in the flow cytometer. Culture supernatants were analyzed for GM-CSF, IL-1beta, and TNF-alpha by ELISA. RESULTS: TSST-1, in a concentration-dependent manner starting at 0.1 pg/mL, significantly inhibited monocyte apoptosis and resulted in the production of the prosurvival cytokines GM-CSF, IL-1beta, and TNF-alpha. In coculture conditions with conditioned media from TSST-1-stimulated monocytes, with or without neutralizing antibody to the various cytokines, the data show GM-CSF production was responsible for the inhibition of apoptosis. CONCLUSIONS: The data strongly suggest that staphylococcal exotoxins known to colonize skin lesions on patients with chronic AD may induce the production of GM-CSF, resulting in inhibition of monocyte-macrophage apoptosis, and thereby contribute to the chronicity of this inflammatory disease.

Bleomycin-induced lung disease in an animal model: correlation between computed tomography-determined abnormalities and lung function.

RATIONALE AND OBJECTIVES: The authors evaluated whether specific types of computed tomographic (CT) abnormalities could be correlated with physiologic impairment in animals with bleomycin-induced lung injury. METHODS: Lung injury was induced in 20 rabbits by means of intratracheal administration of bleomycin (3 U per kilogram of body weight), followed by 100% oxygen for 2 minutes. The animals underwent high-resolution CT scanning at 14 (n = 4), 28 (n = 6), or 56 (n = 10) days after injury. CT morphometry was used to determine the extent of abnormal lung. Physiologic evaluation was performed before injury and before scanning. RESULTS: The overall extent of abnormal lung and of parenchymal opacification on CT scans did not correlate with any physiologic variable. The extent of interstitial thickening correlated significantly with total lung capacity (r = -.783, P = .0005), airway pressure at maximal lung volume (r = .836, P = .0001), and alveolar-arterial oxygen gradient (r = .613, P = .004). CONCLUSION: CT findings of interstitial thickening are associated with impaired gas exchange and lung stiffness in rabbits.

A novel action of IL-13: induction of diminished monocyte glucocorticoid receptor-binding affinity.

We have recently demonstrated that the combination of IL-2 and IL-4 blunts T cell responses to glucocorticoids in steroid resistant (SR) asthma by reducing glucocorticoid receptor (GCR)-binding affinity. Since immune activation appears to be involved in the acquisition of steroid resistance, we sought to identify whether other cytokines could also induce diminished GCR-binding affinity. In the current report, utilizing a [3H]dexamethasone radioligand-binding assay and Scatchard analysis, we found that IL-13, a cytokine with similar actions as IL-4, could induce diminished GCR binding-affinity (GCR Kd = 34.4 +/- 2.3 nM with IL-13 vs Kd = 8.8 +/- 0.7 nM for unstimulated control cells; p < 0.001) in PBMC from normal subjects. In contrast, PBMC incubated with IL-1, IL-3, IL-5, IL-7, IL-8, IL-12, or granulocyte-macrophage-CSF had no effect on GCR-binding affinity; and no additive effect to the decreased GCR-binding affinity was noted when IL-13 was cocultured with IL-2 or IL-4. The cell target of IL-13-induced GCR effects was studied and found to reside in the non-T cell population; specifically, the monocyte fraction. To determine the functional significance of the decreased GCR-binding affinity, monocytes were pretreated with and without IL-1 3 prior to stimulation with LPS and hydrocortisone. IL-13 pretreatment of monocytes significantly diminished (p = 0.005) the suppressive effects of hydrocortisone on LPS-induced IL-6 production. IL-13, by virtue of its ability to induce diminished GCR-binding affinity, may contribute to impaired GC responsiveness during inflammatory illnesses.

Granulocyte macrophage colony-stimulating factor contributes to enhanced monocyte survival in chronic atopic dermatitis.

Evidence suggesting that prolonged effector cell survival may contribute to perpetuation of inflammation prompted us to ask whether monocyte macrophages, the predominate inflammatory cell in the lesion of chronic atopic dermatitis (AD), exhibit enhanced survival in AD. Cultures of peripheral blood monocytes from patients with chronic AD, psoriasis, and from normal (NL) donors were examined for morphologic features and DNA fragmentation characteristic of cells undergoing the process of apoptosis (programmed cell death). Cultures of AD monocytes exhibited a significantly lower incidence of apoptosis than did cultures of NL monocytes (45 vs 68%, P < 0.01), or psoriatic monocytes (45 vs 80%, P < 0.01). Furthermore, AD monocytes were unresponsive to both IL-1, an inhibitor of apoptosis, and IL-4, an enhancer of apoptosis, in comparison to cultured NL monocytes. Of note, GM-CSF in a concentration-dependent fashion, decreased the incidence of apoptosis in NL monocyte cultures and rendered them unresponsive to these cytokines. These findings suggested that GM-CSF may enhance monocyte survival in AD. In support of this hypothesis, AD monocyte cultures produced fivefold more GM-CSF than did cultures of NL monocytes or psoriatic monocytes (P < 0.05). Additionally, there was a significantly greater number of GM-CSF mRNA expressing cells detected by in situ hybridization in biopsies of lesions of chronic AD than in acute AD or NL skin (P < 0.05). Finally, NL monocytes incubated with supernatants obtained from monocytes of AD patients exhibited significant inhibition of apoptosis, an effect that could be ablated by a neutralizing antibody to GM-CSF. Taken together, these data strongly suggest that increased production of GM-CSF by cells from patients with AD inhibits monocyte apoptosis and may contribute to the chronicity of this inflammatory disease.

Lipolysaccharide-induced monocyte retention in the lung. Role of monocyte stiffness, actin assembly, and CD18-dependent adherence.

Blood monocytes and monocyte-derived macrophages accumulate in the lungs and can modulate pulmonary inflammatory and reparative processes through their elaboration of cytokines and growth factors. Endotoxemia, often a prelude to acute lung injury, induces a monocytopenia, likely resulting from monocyte accumulation in the lung. We hypothesized that LPS would induce monocyte lung retention by increasing monocyte stiffness and thereby diminishing the cell's ability to deform and transit the narrow pulmonary capillary network, and that LPS would induce CD18-dependent adhesion of monocytes to endothelium, prolonging their retention. LPS induced a rapid and concentration-dependent increase in human monocyte stiffness, net filamentous actin assembly, and retention in a filtration model of pulmonary capillaries. These LPS-induced responses were dependent on the integrity of actin filaments in that cytochalasin D, an agent that disrupts filamentous actin assembly, attenuated each of these processes. LPS induced CD18-dependent and -independent human monocyte adhesion to unstimulated human endothelial cell monolayers. In vivo, rabbit monocytes were retained in the lungs of animals rendered endotoxemic. Pretreatment of monocytes ex vivo with LPS enhanced their lung retention suggesting that LPS was acting directly on monocytes. Initial lung retention during endotoxemia was attenuated by inhibiting monocyte F-actin assembly with cytochalasin D. Anti-CD18 Abs caused a slight decrease in initial retention of monocytes, but led to a 90% inhibition of retention by 2 h. Control IgG had no effect. These data suggest that the initial retention of monocytes in the lung during endotoxemia is dependent on alterations in their stiffness and assembly/organization of F-actin, and that CD18-dependent adhesive mechanisms prolong monocyte retention in the lung during this process.

Correlation between high resolution computed tomography and tissue morphometry of the lung in bleomycin-induced pulmonary fibrosis in the rabbit.

It is generally considered that the hazy increased density and consolidation seen on high resolution lung CT (HRCT) scans in patients with diffuse interstitial lung disease reflect tissue inflammation, whereas a predominance of linear structures corresponds to tissue fibrosis. The purpose of this study was to determine whether abnormalities observed by HRCT in the lungs of bleomycin-treated rabbits correlated with specific pathologic abnormalities seen on morphometric analysis of lung tissue. Bleomycin was instilled into the lungs of intubated rabbits and followed by inhalation of 100% oxygen. In different animals on Days 14, 28, or 56 after bleomycin, 1 or 1.5 mm HRCT scans were obtained at 8-mm intervals. Subsequently, the right lungs were processed for histology, and the left lungs were processed for determination of hydroxyproline content. Using a morphometric technique, the volume density of normal lung parenchyma, hazy increased density, consolidation, nodules, and central or peripheral lines was determined on each HRCT scan. After the rabbits were killed, the volume density of normal lung parenchyma, intra-alveolar cells, intra-alveolar amorphous material, and thickened interstitium (cellular or acellular) was also determined morphometrically in multiple lung tissue sections in each rabbit. There was a correlation between the volume densities of consolidation on HRCT scans and intra-alveolar cells and amorphous material on tissue morphometry (r = 0.90, p = 0.0001) over the 56-day period after bleomycin/oxygen administration. There was no correlation between the volume density of hazy increased lung density (HRCT) and the volume density of these same or any other individual or combined parameters (tissue morphometry) over the 56 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells.

One of the key features associated with programmed cell death in many tissues is the phagocytosis of apoptotic bodies by macrophages. Removal of apoptotic cells occurs before their lysis, indicating that these cells, during the development of apoptosis, express specific surface changes recognized by macrophages. We have compared the mechanisms by which four different macrophage populations recognize apoptotic cells. Murine macrophages elicited into the peritoneal cavity with either of two different phlogistic agents were able to phagocytose apoptotic cells. This phagocytosis was inhibited by phosphatidylserine (PS), regardless of the species (human or murine) or type (lymphocyte or neutrophil) of the apoptotic cell. In contrast, the murine bone marrow macrophage, like the human monocyte-derived macrophage, utilized the vitronectin receptor, an alpha v beta 3 integrin, for the removal of apoptotic cells, regardless of their species or type. That human macrophages are capable, under some circumstances, of recognizing PS on apoptotic cells was suggested by the observation that PS liposomes inhibited phagocytosis by phorbol ester-treated THP-1 cells. These results suggest that the mechanism by which apoptotic cells are recognized and phagocytosed by macrophages is determined by the subpopulation of macrophages studied.

Mechanisms of lipopolysaccharide-induced neutrophil retention. Relative contributions of adhesive and cellular mechanical properties.

Intravascular LPS rapidly induces neutrophil sequestration in pulmonary capillaries by mechanisms that, although currently unknown, must take into account the size difference between the neutrophil and capillary diameter. To determine whether LPS alters neutrophil stiffness, and hence the ability of neutrophils to traverse capillaries, neutrophil passage through pulmonary capillaries was modeled by passage through filters with 6.5-microns pores. LPS increased retention in the pores in a concentration-dependent fashion that required the presence of heat-inactivated platelet-poor plasma, and was evident as early as 10 min after stimulation. The effect of LPS on the structural properties of the neutrophil was then studied. LPS induced f-actin reorganization in neutrophils in the presence of plasma. Disruption of actin organization and assembly with cytochalasin D completely inhibited early LPS-induced retention and attenuated retention at later timepoints, indicating that LPS-stimulated retention depends on filament organization. LPS-induced actin assembly and retention were abrogated by an antibody directed against CD14, a putative LPS receptor. CD18-dependent adherence of neutrophils contributed significantly to retention only at later timepoints with no significant contribution to retention at 20 min as determined by inhibition of adherence with the mAb 60.3. Morphometric assessment of neutrophil accumulation in the lungs of rabbits given 1 microgram LPS showed a marked increase in apparent neutrophil number, which was unaltered by antibodies to CD18, suggesting that mechanisms other than adhesion may account for accumulation in vivo. Direct measurements showed that neutrophil stiffness increased with exposure to LPS in a fashion similar to LPS-induced retention and actin organization. Pretreatment of neutrophils with cytochalasin D attenuated the increased stiffness. These data suggest that reorganization of filamentous-actin induced by LPS leads to cell stiffening and retention in capillary-sized pores. Although the organization of f-actin continues to be important in retention at later time points, adherence of cells also contributes significantly to cell retention. The changes in mechanical properties of the neutrophil may be important in the sequestration of neutrophils in pulmonary capillaries noted in endotoxemia.

Prolonged monocyte accumulation in the lung during bleomycin-induced pulmonary fibrosis. A noninvasive assessment of monocyte kinetics by scintigraphy.

It has become more evident that monocytes, macrophages, and their products interact in a complex manner with various cell types in the lung, and may under the proper set of conditions contribute to the pathogenesis of pulmonary fibrosis. Current methods used to assess the lung content of mononuclear cells, which include tissue immunohistochemistry and bronchoalveolar lavage fluid analysis, sample the lung at one point in time and therefore provide only a "snapshot" of dynamic process. We utilized external imaging (scintigraphy) to provide a dynamic assessment of the trafficking patterns of radiolabeled monocytes in the lungs of rabbits in conjunction with lung tissue morphometry and bronchoalveolar lavage fluid analysis to determine the kinetics of neutrophil and monocyte accumulation in the alveolar walls and alveolar spaces of the lung during bleomycin-induced pulmonary fibrosis. We found that scintigraphy accurately reflected the accumulation of monocyte-associated radioactivity in the alveolar walls over time as well as the subsequent migration of these cells into alveolar spaces during the acute phase of bleomycin-induced lung injury (days 0 to 14) when compared with lung tissue morphometry. The scintigraphy, lavage, and morphometry data together showed that neutrophil influx into both of these lung compartments preceded that of monocytes by days, and that the influx of monocytes accounted for a major proportion of mononuclear cells found in the alveolar walls and alveolar spaces of the lung during this acute phase of inflammation. The increased numbers of neutrophils and mononuclear cells in alveolar spaces normalized by days 14 and 28 respectively, but in contrast to the normalization of neutrophil content in alveolar walls by day 10, increased numbers of mononuclear cells persisted in alveolar walls for up to 56 days, a time when there was a significant increase in the hydroxyproline content of these lungs. These data also show that the increased number of mononuclear cells present in the alveolar walls on days 28 and 56 was not due to a persistent influx of blood monocytes. These data suggest: (a) that differential pathways of efflux existed for alveolar wall versus alveolar space mononuclear cells, (b) that a delay in efflux from the alveolar walls occurred and/or that an increase in the local proliferation of mononuclear cells in this compartment may have been occurring during the later phases of bleomycin-induced lung injury, and (c) that this prolonged residence of mononuclear cells in the alveolar walls occurred concurrently with the development of pulmonary fibrosis.

Adherence-dependent increase in human monocyte PDGF(B) mRNA is associated with increases in c-fos, c-jun, and EGR2 mRNA.

Adherence is an important initial step in the transition of a circulating monocyte to a tissue macrophage. This differentiation is accompanied by an augmented capacity to generate growth factors. We hypothesized that adherence itself might be an important trigger for a sequence of gene activation culminating in cells with increased mRNA encoding profibrotic growth factors such as platelet-derived growth factor B subunit (PDGF[B]) and transforming growth factor-beta (TGF-beta). After in vitro adherence, human monocytes had a biphasic increase in PDGF(B) mRNA with peaks at 6 h and 13 d. No increase in TGF-beta mRNA was observed. The 6-h increase in PDGF(B) mRNA was adherence dependent, and in addition, was abrogated when the cytoskeletal integrity was compromised by cytochalasin D. The 6-h increase in PDGF(B) mRNA was unaltered by adherence in the presence of the monocyte stimulus lipopolysaccharide. Adherence to either fibronectin or collagen-coated plastic had little consistent effect on PDGF(B) mRNA accumulation. The increased PDGF(B) mRNA observed in adherent monocytes was accompanied by increases in mRNAs of the early growth response genes c-fos (maximal at 20 min), c-jun, and EGR2 (maximal at 6-24 h). The increase in c-jun and EGR2, but not c-fos, mRNA was also abrogated by cytochalasin D. These observations suggest that adherence results in increases of c-fos, c-jun, EGR2, and PDGF(B) mRNA. In addition, the increases in c-jun, EGR2, and PDGF(B) may depend on cytoskeletal rearrangement. Modulation of these events at the time of adherence offers a mechanism by which differential priming of the cells may be accomplished.

Retention of leukocytes in capillaries: role of cell size and deformability.

Leukocytes within the circulation are in dynamic equilibrium with a marginated pool, thought to reside mainly within the pulmonary capillaries. The size discrepancy between the mean diameter of circulating leukocytes (6-8 microns) and that of the pulmonary capillaries (approximately 5.5 microns) forces the cells to deform in order to transit the capillary bed. Consequently, we investigated the hypothesis that the biophysical properties of cell size and deformability determined differential leukocyte retention in the lung. Comparison of the filtration properties of human neutrophils, lymphocytes, monocytes, platelets, and erythrocytes through polycarbonate filters (5-micron pore diameter) revealed that the largest leukocytes (neutrophils and monocytes) were retained to the greatest extent and the smaller cells (lymphocytes and platelets) the least. Undifferentiated HL-60 cells, of greater diameter than their differentiated counterparts, were also retained to a greater extent, confirming that cell size was one important determinant of retention in these model capillaries. However, compared with neutrophils, which are of similar diameter, monocytes were retained to a greater extent, suggesting that monocytes might be less deformable than neutrophils. To test this hypothesis, deformability was measured directly using the cell poker. Monocytes were found to be the stiffest, neutrophils the softest, and lymphocytes intermediate. Glutaraldehyde treatment of neutrophils markedly increased their stiffness and decreased their ability to transit the pores of the filters in vitro and the pulmonary microvasculature of rabbits without changing their adhesive properties or size. These observations support the hypothesis that biophysical properties of leukocytes (size and deformability) determine in part their ability to transit the pulmonary capillaries and may determine the magnitude of their marginated pools.

Fibronectin fragments containing the RGDS cell-binding domain mediate monocyte migration into the rabbit lung. A potential mechanism for C5 fragment-induced monocyte lung accumulation.

Many inflammatory processes are characterized by an early phase of neutrophil migration and a later phase of monocyte migration into the inflammatory site. Mechanisms that govern the transition between phases are the subject of these investigations. Acute lung inflammation induced by C5 fragments in the rabbit leads to an initial neutrophil influx and plasma leakage into the alveolar space, followed by monocyte influx that we have previously shown to be dependent on prior emigration of neutrophils. Neutrophil enzymes are known to cleave intact fibronectin into fragments that are monocyte chemotaxins in vitro. Accordingly, generation of appropriate fibronectin fragments in situ by proteolytic enzymes from infiltrating neutrophils might represent a potential mechanism for attraction of monocytes into the lung. The studies reported herein demonstrate that a 120-kD fragment of fibronectin containing the RGDS fibroblast cell-binding domain induced monocyte migration into the rabbit lung in vivo. Intact fibronectin was inactive. A significant proportion of the monocyte migration was neutrophil independent. Intact fibronectin was present in bronchoalveolar lavage fluid from C5 fragment-treated animals rendered neutropenic, but absent in lavage from normal C5 fragment-treated animals. Fibronectin fragments were present in bronchoalveolar lavage fluid from both C5 fragment-treated and control rabbits. In addition, the amount of fibronectin was significantly increased in lavage of C5 fragment-treated normal but not neutropenic animals. Monoclonal antibodies directed against an epitope of fibronectin containing the RGDS cell-binding domain significantly inhibited the C5 fragment-induced monocyte migration, but not neutrophil migration. These studies suggest that chemotactic fibronectin fragments may in part be responsible for the recruitment of monocytes into areas of acute lung inflammation.

Leukocyte and platelet margination within microvasculature of rabbit lungs.

These studies compare the behavior of radiolabeled neutrophils, monocytes, lymphocytes, and platelets during their first pass through the pulmonary circulation after a central venous injection and their distribution within the circulation 10 min later. Their first pass through the pulmonary circulation was compared with erythrocytes (RBCs) using the indicator-dilution technique, and their recovery within the circulation of the lung and other organs was determined at 10 min by counting the radioisotopes in each organ. The extraction of each cell relative to RBCs during the first pass through the lung correlated with cell size in that the neutrophils (volume 107-140 fl) showed 97.6 +/- 0.6% extraction, monocytes (volume 80-105 fl) showed 91.4 +/- 1.7% extraction, lymphocytes (volume 36-75 fl) showed 80.1 +/- 4.4% extraction, and platelets (volume 4-7 fl) showed 33.1 +/- 3.9% extraction. After 10 min of circulation, the proportion of injected cells remaining in the lung was similar for neutrophils and monocytes (27.4 +/- 1.8 vs. 31.4 +/- 1.6%) but lower for lymphocytes (18.6 +/- 2.9%) and platelets (3.1 +/- 0.5%). All of the leukocytes were found to have a substantial marginated pool within the lung, whereas the platelets did not. The exchange between the circulating and marginated pools of leukocytes in the lung was related to blood velocity, with the least retention occurring in lung regions with shortest RBC transit times. We conclude that cell size is a major factor determining the time that cells will be delayed by the pulmonary microvasculature.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipopolysaccharide stimulates monocyte adherence by effects on both the monocyte and the endothelial cell.

The effects of the LPS moiety of endotoxin on monocyte adherence to an endothelial cell surface were investigated over times before the development of well described LPS-induced endothelial cell surface adhesive molecules. In an in vitro microtiter adherence assay, LPS in concentrations of 10 ng/ml to 10 micrograms/ml incubated for 20 to 60 min with human monocytes significantly stimulated monocyte adherence to human umbilical vein endothelial cell monolayers (HUVEC) and serum-coated plastic surfaces. The time course and concentration dependence of LPS-stimulated monocyte adherence to glutaraldehyde-fixed HUVEC did not differ significantly from that to unfixed HUVEC or serum-coated plastic surfaces. Pretreatment studies suggested that LPS acted on the monocyte within 25 min to stimulate adherence to untreated endothelial cells but required a minimum of 1.5 to 2 h to render the endothelial cell more adhesive for untreated monocytes. The potential role of TNF-alpha, IL-1 alpha, and IL-1 beta in this system was assessed by determining the ability of these cytokines (+/- cytokine antibodies) to increase monocyte adherence. TNF, but neither IL-1, stimulated early monocyte adherence (1 h). This TNF-stimulated monocyte adherence was abrogated by coincubation with anti-rTNF-alpha polyclonal antibody. However, the anti-rTNF antibody had no effect on LPS-induced monocyte adherence to endothelial cells or serum-coated plastic surfaces. An early action of LPS on the monocyte to induce adherence to endothelial cell surfaces may contribute to the initial localization of peripheral blood monocytes in tissues during endotoxemia. The later effects of LPS on the endothelial cell to stimulate monocyte adherence may then amplify these initial monocyte-endothelial cell interactions to prolong and intensify monocyte adherence prior to migration into tissues.

Monocyte retention and migration in pulmonary inflammation. Requirement for neutrophils.

The acute inflammatory process is characterized by an orderly progression of events; an initial phase of early neutrophil accumulation and a later phase of mononuclear cell (including monocyte) accumulation. The mechanisms which control the transition from one phase to the other are largely unknown. We present a rabbit model of C5 fragment (C5f)-induced lung inflammation in which purified radiolabeled peripheral blood neutrophils and monocytes were used as probes to monitor the retention and emigration of these leukocytes into well localized areas of inflammation. Neutrophil preparations (greater than 95% pure) were isolated by discontinuous plasma-Percoll density gradients, and monocyte preparations (greater than 91% pure) were isolated by counterflow cell elutriation, labeled with 111Indium-tropolonate, and intravenously infused into separate recipient animals. The monocytes circulated with a half-life of approximately 30 hours. The retention of labeled monocytes or neutrophils within the lung was monitored scintigraphically. C5f-induced monocyte lung retention was delayed 2 to 4 hours compared with neutrophil lung retention. Radiolabeled neutrophils were selectively retained in the area of C5f-induced inflammation (right cranial lung lobe, RCL) as early as 20 minutes after the induction of the inflammatory response, reached a maximum by 2 hours, and were not retained by 48 hours after C5f instillation. The signal inducing C5f-induced monocyte lung retention was shown to be transient. Monocytes were selectively retained in the RCL if the area of inflammation was induced 2 to 4 hours but not 15 minutes or 16 hours before their infusion. The time course of C5f-induced monocyte migration into the alveolar space determined by lavage analysis was delayed 2 to 3 hours compared with neutrophil migration. Neutrophils selectively migrated into the RCL 1 to 2 hours after the induction of the inflammatory response, reached a maximum by 4 hours, and had disappeared by 48 hours. Radiolabeled monocytes selectively migrated into the RCL 3 to 4 hours after the induction of the inflammatory response, reached a maximum by 4 hours, and remained present through 48 hours. The total number of labeled and unlabeled mononuclear cells present in the C5f-treated RCL lavage at 48 hours was significantly increased above controls. The signal for this monocyte migration (as for lung retention) was shown to be transient in that radiolabeled monocytes did not migrate when infused 16 hours after the induction of the inflammatory response. C5f did not induce monocyte lung retention nor monocyte migration into the alveolar space of animals rendered neutropenic.(ABSTRACT TRUNCATED AT 400 WORDS)