GM-CSF expression by human lung microvascular endothelial cells: in vitro and in vivo findings

Jürgen Burg, Vera Krump-Konvalinkova, Fernando Bittinger, and Charles James Kirkpatrick

Institute of Pathology, Johannes Gutenberg University, 55101 Mainz, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recently, many findings indicate that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the pathogenesis of acute and chronic lung diseases. In the present paper, the production of this cytokine in human pulmonary microvascular endothelial cells (HPMEC) is investigated. In an in vitro study, quiescent HPMEC did not express GM-CSF, either at the transcriptional or at the protein level. After activation for 4 h with tumor necrosis factor (TNF)-alpha (30/300 U/ml), lipopolysaccharide (LPS; 0.1/1 µg/ml), or interleukin (IL)-1beta (100 U/ml), a significant release of GM-CSF was measured by enzyme-linked immunosorbent assay, with a time-dependent increase over 72 h. IL-8 (4, 16, or 64 ng/ml) or IL-1beta at a concentration of 10 U/ml did not induce the release of GM-CSF. Human umbilical vein endothelial cells (HUVEC) and the angiosarcoma cell line HAEND served as reference cell lines. GM-CSF release in HPMEC was significantly (P < 0.025-0.05) less inducible by IL-1beta than in HUVEC. A constitutive expression of GM-CSF by HAEND was observed. Additionally, GM-CSF expression in vivo by the lung microvasculature was confirmed by immunohistochemistry in lung tissue. To our knowledge, this is the first report of the ability of human pulmonary endothelial cells to synthesize and release GM-CSF. These results support the hypothesis that the lung microvasculature via the production of GM-CSF is a potential contributor to the cytokine network in lung diseases. This could be of particular importance in the pathogenesis of the acute respiratory distress syndrome in which endothelial dysfunction plays a central pathogenetic role.

granulocyte-macrophage colony-stimulating factor; human lung; microvasculature; endothelium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BESIDES HEMODYNAMIC AND hemostatic functions, the microvasculature of the lung is involved in the recruitment of inflammatory blood cells into the interalveolar space and is therefore crucial in both acute and chronic lung diseases (6, 35, 47, 48,). Apart from these more permissive functions, little is known about the direct contribution of the microvascular endothelium to lung pathogenesis.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), which was originally identified because of its effects on hematopoetic progenitors, has proved to exert varying effects on fully differentiated cells. The human GM-CSF gene is located on chromosome 5 in the same region as interleukin (IL)-3, IL-4, IL-5 and the macrophage colony-stimulating factor receptor. The human GM-CSF protein contains 144 amino acid residues, including a 17-amino-acid signal peptide, which is removed during secretion. Native GM-CSF is heavily glycosylated, resulting in a molecular mass of 14.5-34 kDa (for review, see Ref. 11). The GM-CSF receptor is a member of the hematopoietin receptor superfamily and is comprised of an alpha -chain (GM-CSF receptor-alpha ) specific for GM-CSF and a beta -chain shared with the IL-3 and IL-5 receptors (24, 30).

Recent findings reported from different laboratories suggest a pivotal role of GM-CSF in the pathogenesis of inflammatory lung diseases, acute respiratory distress syndrome (ARDS), and lung fibrosis. In asthma, enhanced GM-CSF production by lung inflammatory cells and bronchial epithelial cells has been well documented (5, 8, 10). There is evidence that GM-CSF is important in regulating pulmonary surfactant homeostasis and that reduced activity of GM-CSF and its receptor is associated with alveolar proteinosis (34, 36, 40, 50). In patients suffering from ARDS, it has recently been shown that GM-CSF is elevated in bronchioalveolar fluid and that GM-CSF has an influence on granulocyte viability (13, 28). Septic preterm infants had significantly higher plasma concentrations of GM-CSF than healthy preterm infants (18). In addition, after intravenous endotoxin challenge in humans, an increase in plasma GM-CSF was observed (23).

There is now a large body of evidence indicating that GM-CSF plays a particular role in the development of lung fibrosis, although reports are conflicting. Xing et al. (43, 44, 45) have shown that overexpression of GM-CSF in the rat lung causes accumulation of eosinophils and macrophages in the early stages, followed by fibrosis in later stages. Overexpression of GM-CSF in type II alveolar cells of transgenic mice increased lung size and caused type II cell hyperplasia, which is associated with acute and chronic lung diseases (16). In bleomycin-induced rat lung fibrosis, total mRNA of GM-CSF in the lung was already upregulated after 6 h and returned to basal levels after 24 h, followed by upregulation of transforming growth factor (TGF)-beta (2), which suggests a particular role in the early phase of pulmonary fibrosis. Piguet et al. (33) observed that the application of a neutralizing antibody to GM-CSF markedly aggravated collagen deposition in bleomycin-induced lung damage in mice. On the other hand, results from Moore et al. (31) support the theory of an anti-fibrotic potential of GM-CSF, since bleomycin-induced lung fibrosis was more marked in GM-CSF knock-out mice. After bleomycin injury, they also observed a diminished expression of GM-CSF in isolated alveolar epithelial cells from rats (7).

Cultured human lung fibroblasts release GM-CSF constitutively, and this cytokine has been shown to exert a chemokinetic effect on monocytes (22). Moreover, there is evidence from in vitro experiments using human umbilical endothelial cells (HUVEC) and human monocytes that direct monocyte-endothelial interaction induces GM-CSF production in both cell types (39). Recently, coculture experiments using IL-1beta -activated HPMEC and human eosinophils have shown that GM-CSF mRNA expression is upregulated in transmigrated eosionophils and that in vitro survival is longer (46).

In humans, inflammatory cells, lung macrophages, and bronchial epithelial cells are capable of producing GM-CSF (1, 3, 10, 21) so that in the complex microanatomy of the lung a variety of the cell types could contribute to GM-CSF levels in various pulmonary diseases.

Little is known about the role of microvascular endothelial cells of the lung in this context. Nevertheless, because of the essential pathogenetic role of the dysfunctional microcirculation in the development of ARDS and multiple organ dysfunction syndrome (19, 26), we investigated GM-CSF expression by primary isolated human pulmonary microvascular endothelial cells (HPMEC), both in the unstimulated state and after pretreatment with proinflammatory stimuli [tumor necrosis factor (TNF)-alpha , IL-1beta , lipopolysaccharide (LPS), and IL-8]. Macrovascular endothelial cells from HUVEC and an angiocarcoma cell line (HAEND) served as reference cell lines. To study the in vivo relevance of these data, the expression of GM-CSF in inflammed lung tissue was also investigated by immunohistochemistry.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Isolation, Culture, and Characterization

HUVEC were isolated according to the method described by Jaffe et al. (17). The cells were cultured in a 1:1 mixture of Ham's F-12 medium and Iscove's modified Dulbecco's medium (GIBCO-BRL) supplemented with 20% FCS, penicillin-streptomycin solution (20 U/ml-20 µg/ml; GIBCO-BRL), and L-glutamine (2 mM; GIBCO-BRL) in a humidified atmosphere containing 5% CO2 and 10% O2.

Isolation and culture of HPMEC were performed by a modification of the method of Hewett and Murray (14) and Kirkpatrick et al. (20). Normal human lung tissue was obtained from lobectomy specimens resected because of lung tumors. No adjuvant chemotherapy had been given. Briefly, isolation of HPMEC was performed as follows: subpleural lung tissue was cut into small fragments with scissors. After removal of debris and erythrocytes through a 40-µm nylon net, the tissue was treated with dispase (1.18 U/ml at 4°C for 18 h). After filtration through a 100-µm nylon net, the tissue was treated in a volume of 4 ml with elastase (40 units), trypsin (0.05%), and EDTA (1.8 mM) for 30 min at 37°C followed by a further 100-µm net filtration. The cell clumps were repeatedly resuspended in PBS-BSA and filtered through a 40-µm net, followed by centrifugation for 10 min and resuspension in medium-199 plus 20% pooled FCS. This mixed cell culture had a purity of ~30 ± 5% concerning the CD31-positive endothelial cells. The culture was cultivated at 37°C in a gas mixture of 5% CO2 in air for 5-7 days, but not longer, to avoid overgrowth of contaminant cells. The developing monolayer was disrupted by treatment with 0.2% EDTA and 0.2% BSA for 30 min at 37°C followed by a mixture of 0.25% trypsin and 0.25% EDTA for 1 min. The positive selection of HPMEC was achieved by interacting the cell suspension with magnetic beads (1 µm diameter; Dianova) coated with a mouse monoclonal antibody against human platelet endothelial cell adhesion molecule-1 (PECAM-1; Immunotech). The subsequent pure cultures of PECAM-1-positive HPMEC were also shown to be positive for CD34, factor VIII-related antigen, CD36 (thrombospondin receptor), Ulex europaeus agglutinin-1, prostacyclin, IL-1, IL-6, and plasminogen activator inhibitor-1. The cells also showed uptake of 1,1'-dioctadecyl-1,3,3',3'-tetramethyl-indocarbocyanine-acetylated low-density lipoprotein (Dil-Ac-LDL). Contaminant cells were detected by immunocytochemistry with antibodies against CD68, cytokeratins, or smooth muscle actin (SMA). Fibroblasts or SMA-negative myofibroblast-like cells were identified because of their characteristic morphology and rapid growth with nest formation. When no contaminat cells were detected, purity was determined at >99%. The viability, growth characteristics, and immunohistochemical phenotype were unchanged until passage 12.

HAEND cells were a gift of Dr. V. Vetvicka (University of Louisville, Kentucky). HAEND cells were originally derived from a human liver angiosarcoma (15). In our laboratory, we confirmed immunohistochemical positivity for von Willebrand factor and Ulex europeus agglutinin-1; uptake of Dil-Ac-LDL was also demonstrated. The cells were cultured in RPMI 1640 supplemented with 10% FCS, penicillin-streptomycin solution (20 U/ml-20 µg/ml; GIBCO-BRL), and L-glutamine (2 mM; GIBCO-BRL).

Activation of Cells, Analysis by Enzyme-Linked Immunosorbent Assay, and RT-PCR

Cell activation. Before activation, the viability of the cells was tested by morphological inspection and Trypan blue exclusion. Cells were seeded in 24-well culture plates and allowed to adhere overnight (125,000 cells/well; 0.625 ml growth medium). Subconfluent cultures of HPMEC (passages 3-7) or HUVEC (passages 2-3) were stimulated by adding TNF-alpha (30/300 U/ml; Sigma), IL-1beta (10/100 U/ml; Strathmann Biotech, Hannover, Germany), LPS (0.1/1 µg/ml; Sigma), or IL-8 (4, 16, or 64 ng/ml; Sigma) to the culture medium for 4, 24, or 72 h.

After the activation period, the supernatant was harvested and stored at -20°C. Supernatant (100 µl) correlated approximately with an original cell count of 20,000. For RNA isolation, cell lysates were stored in TRIzol (Life Technologies). The activation experiments with HUVEC and HPMEC were each repeated with four different donors.

HAEND cells were activated after 4 and 24 h with LPS (1 µg/ml), TNF-alpha (300 U/ml), and IL-1beta (100 U/ml). These experiments were repeated three times.

Enzyme-linked immunosorbent assay analysis. The concentration of GM-CSF in the supernatant was measured at three different dilutions (1:1, 1:8, and 1:16) by a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Quantikine; R&D Systems). Each dilution was measured in triplicate. Samples were quantitated from the linear portion of the standard curve, with detection limits between 3 and 5 pg/ml.

RT-PCR analysis. Total cellular RNA of unstimulated HPMEC and HUVEC or after 24 h of stimulation with LPS (1 µg/ml) was prepared using TRIzol reagent according to the instructions of the manufacturer (Life Technologies). First-strand cDNA synthesis was performed using the Superscript Preamplification System (Life Technologies) according to the manufacturer's instructions. Two micrograms of total cellular RNA were reverse transcribed using random hexamers. The reaction products were diluted to 200 µl to be within the linear range of PCR amplification. Two-microliter aliquots of diluted cDNAs were used in PCR amplifications. Amplification reactions were run in a Perkin-Elmer 2400 thermocycler. PCR conditions for GM-CSF and platelet-derived growth factor (PDGF)-alpha were as follows: denaturation for 2 min at 94°C followed by 35 three-step amplification cycles of 94°C for 45 s, 55°C for 45 s, followed by a final extension step at 72°C for 8 min. PCR conditions for beta -actin were denaturation for 2 min at 94°C followed by 35 three-step amplification cycles at 94°C for 45 s, 65°C for 45 s, 72°C for 45 s, and a final extension step at 72°C for 8 min. The PCR reaction mixtures (10 µl) were analyzed on a 2% agarose gel containing ethidium bromide. The oligonucleotide primer pairs were designed to preferentially amplify cDNA and to allow distinction between genomic and cDNA amplification products. The sequences of primers and length of PCR products were as follows: GM-CSF (190 bp): sense, 5'-CTG CTG CTG AGA TGA ATG AAG-3' and antisense, 5'-GCA CAG GAA GTT TCC GGG GT-3'; PDGF-alpha (225 bp): sense, 5'-CCT GCC CAT TCG GAG GAA GAG-3' and antisense, 5'-TTG GCC ACC TTG ACG CTG CG-3'; and beta -actin (574 bp): sense, 5'-GAC CTG ACT GAC TAC CTC ATG A-3' and antisense, 5'-AGC ATT TGC GGT GGA CGA TGG AG-3'.

Tissue Preparation and Immunohistochemistry

Lung tissue was obtained from peritumoral pneumonia, from two cases of lung explantation because of end-stage cystic fibrosis, and chronic candida pneumonia with sepsis. Uninflammed lung tissue served as a control and was obtained from a lung lobe unaffected by tumor. Small tissue fragments were fixed in an ethanol-based medium (Notox; Quartett) for 4 h taken through graded alcohols to 100% alcohol for 3 h and then routinely embedded in paraffin. Paraffin sections (4 µm thick) were stained using the avidin-biotin complex (ABC) method. After blocking unspecific avidin or biotin binding with a blocking kit (Avidin/Biotin Blocking Kit; Vector Laboratories, Burlingame, CA), nonspecific binding of immunoglobulins was blocked by preincubation with 4% nonfat dried milk/2% normal rabbit serum (Vector Laboratories) in Tris buffer (pH 7.6) at room temperature for 30 min. Primary polyclonal antibody against GM-CSF (sc-1321; Santa Cruz Biotechnology) was diluted 1:100 in a blocking reagent (Boehringer-Mannheim, Mannheim, Germany). Primary antibody and negative controls were incubated overnight at 4°C. Slides were then rinsed with Tris buffer (pH 7.6) and incubated for 30 min at room temperature with a secondary rabbit anti-goat antibody in a dilution of 1:200 (BA-5000; Vector Laboratories).

Incubation with the ABC complex conjugated with alkaline phosphatase (DAKO, Hamburg, Germany) for 30 min at room temperature was followed by the addition of substrate using the new fuchsin method. Endogenous alkaline phosphatase was blocked by adding levamisole (Sigma) to the substrate solution. Color development was stopped by immersing the slides in Tris buffer (pH 7.6). Counterstaining was performed with hematoxylin, and the slides were mounted with glycerin gelatine (Merck, Darmstadt, Germany).

Negative controls were incubated with an isotype control (normal goat IgG, sc 2028; Santa Cruz Biotechnology) and showed no reactivity.

Statistical Analyses

Data were statistically evaluated using the Mann-Whitney U-test. Statistical analyses were performed with the help of Microsoft Excel 97. P values <0.05 were taken as statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Analysis of GM-CSF Expression in HPMEC by ELISA and RT-PCR

Quiescent HPMEC neither transcribe nor release GM-CSF. Analysis of supernatants of unstimulated HPMEC, cultivated in fresh growth medium for 4, 24, and 72 h, was performed. The data from the ELISA technique indicate that the cells did not release measurable amounts of GM-CSF. This observation was confirmed with HPMEC from different donors. Furthermore, RT-PCR of total cellular RNA from unstimulated HPMEC showed no constitutively expressed GM-CSF transcript, in contrast to beta -actin (a housekeeping gene) and PDGF-alpha , which were both transcribed constitutively (Fig. 1).


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Fig. 1.   RT-PCR analysis of total cellular RNA. No transcript of granulocyte-macrophage colony-stimulating factor (GM-CSF) was detectable in quiescent human pulmonary microvascular endothelial cells (HPMEC), but, after activation with lipopolysaccharide (LPS; 1 µg/ml) for 24 h, a strong signal at 190 bp is detected, indicating the transcript of GM-CSF. Constitutive expression of beta -actin (574 bp) and platelet-derived growth factor (PDGF)-alpha (225 bp) is observed. o, PCR control (PCR amplification without cDNA template); +, lipopolysaccharide (LPS; 1 µg/ml) stimulation for 24 h; -, no LPS treatment.

Activation of HPMEC with TNF-alpha , IL-1beta , and LPS led to the expression of GM-CSF. Primary cell isolates were activated with TNF-alpha (300 U/ml), LPS (1 µg/ml), and IL-1beta (100 U/ml) for 4, 24, and 72 h (Fig. 2A). To study the dose-response relationship, 10 times lower concentrations of TNF-alpha , LPS, and IL-1beta were applied (Fig. 2B).


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Fig. 2.   Concentration of GM-CSF (pg · ml-1 · 2 × 10-5 cells) measured by enzyme-linked immunosorbent assay (ELISA) in cell culture supernatants from human umbilical vein endothelial cells (HUVEC) and HPMEC from 4 different donors after 4, 24, and 72 h of activation with tumor necrosis factor (TNF)-alpha (300 U/ml), interleukin (IL)-1beta (100 U/ml), and LPS (1 µg/ml) in A or TNF-alpha (30 U/ml), IL-1beta (10 U/ml), and LPS (0.1 µg/ml) in B. No GM-CSF was detectable in quiescent HPMEC or HUVEC (data not shown). * P < 0.05 and ** P < 0.025, amount of GM-CSF released by HPMEC after activation with IL-1beta is significantly lower compared with HUVEC. Ordinate with concentration of GM-CSF is logarithmic. Data are presented as means ± SD from 4 separate experiments (different patient donors).

In contrast to quiescent HPMEC, after 24 h of activation with LPS (1 µg/ml), analysis of total cellular RNA revealed a strong signal at 190 bp, corresponding to the transcript of GM-CSF (Fig. 1).

After 4 h of stimulation with TNF-alpha (300 U/ml), GM-CSF was released in the supernatant. After 24 h, a marked increase in the expression was observed followed by a further increase after 72 h (Fig. 2A). A 10 times lower concentration of TNF-alpha (30 U/ml) also induced the time-dependent increased expression of GM-CSF, with concentrations being proportionally lower compared with the 10 times higher activation dose (Fig. 2).

The strongest stimulus for HPMEC to produce GM-CSF was LPS (1 µg/ml), with maximal concentrations measured after 3 days, corresponding to 1,305 ± 104 pg · ml-1 · 2 × 10-5 cells. Pretreatment with 0.1 µg/ml LPS led to the expression of GM-CSF in a concentration range only slightly lower than that induced by the 1 µg/ml concentration (Fig. 2B).

As was the case with TNF-alpha and LPS, IL-1beta (100 U/ml) also caused a time-dependent release of GM-CSF over the time period from 4 to 72 h (Fig. 2). However, at a concentration of 10 U/ml, IL-1beta did not induce a measurable GM-CSF production in HPMEC after 4, 24, and 72 h of stimulation (Fig. 2). Three different concentrations of IL-8 (4, 16, or 64 ng/ml) did not induce any release of GM-CSF by HPMEC after 4, 24, or 72 h.

GM-CSF Expression in HUVEC and HAEND Compared With HPMEC

GM-CSF expression in the unstimulated state and after activation with proinflammatory stimuli was different in various cells of endothelial origin. As was the case for HPMEC, HUVEC also showed no constitutive expression of GM-CSF at the protein level or the transcriptional level, as was demonstrated by RT-PCR. Like HPMEC, a time-dependent increase in the production of the cytokine after 4, 24, and 72 h of HUVEC stimulation with TNF-alpha (300 U/ml), IL-1beta (100 U/ml), and LPS (1 µg/ml) was measured in the supernatant (Fig. 2A). Additionally, with 10 times lower concentrations of TNF-alpha , IL-1beta , and LPS a strong response was observed even after 4 h.

HPMEC responded in a much weaker fashion to IL-1beta (100 U/ml) than did HUVEC, as was confirmed with endothelial cells from four different lung donors (Fig. 2A). The difference was statistically significant, as was evaluated by the U-test (P < 0.05 after 4 h, P < 0.025 after 24 or 72 h). To compensate for different levels of response because of the primary nature of the endothelial cells, concentrations of GM-CSF released by IL-1beta (100 U/ml) were related to the concentration of GM-CSF induced by LPS (1 µg/ml) or TNF-alpha (300 U/ml) in each experiment. Again, the difference was found to be significant (P < 0.025-0.05). Moreover, HPMEC in contrast to HUVEC did not respond to IL-1beta at a concentration of 10 U/ml (Fig. 2B).

After activation with TNF-alpha (30 and 300 U/ml), the mean concentration of GM-CSF released by HUVEC was higher than that by HPMEC, especially with the lower concentration, although differences were not statistically significant (Fig. 2). After activation with LPS (0.1 and 1 µg/ml) again no significant difference between the amount of GM-CSF released by HPMEC or HUVEC was observed.

In contrast to HPMEC or HUVEC, the angiosarcoma cell line HAEND exhibited a constitutive expression of GM-CSF (Fig. 3). The expression of GM-CSF was also upregulated by TNF-alpha (300 U/ml), LPS (1 µg/ml), or IL-1beta (100 U/ml), with a marked time-dependent increase (Fig. 3).


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Fig. 3.   Concentration of GM-CSF (pg · ml-1 · 2 × 10-5 cells) in the cell culture supernatants of the angiosarcoma cell line HAEND. Constitutive expression of GM-CSF is observed. Activation of HAEND with TNF-alpha (300 U/ml), LPS (1 µg/ml), and IL-1beta (100 U/ml) for 4 and 24 h led to an enhanced release of GM-CSF. Data are presented as means ± SD for n = 3 experiments.

GM-CSF Production by the Human Lung Microvasculature Was Confirmed by Immunohistochemistry

Lung tissue excised from inflammed peritumoral areas (Fig. 4A), from a candida pneumonia with sepsis (Fig. 4B), and from a lung with end-stage cystic fibrosis demonstrated strong focal positivity for GM-CSF in the microvasculature in areas with florid inflammation (Fig. 4).


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Fig. 4.   GM-CSF expression in lung tissue demonstrated by immunohistochemistry [avidin-biotin complex (ABC)-new fuchsin method]. Peritumoral inflammed lung tissue (A, 1:300) and an area of hemorrhage in the case of candida pneumonia with sepsis (B, 1:800) exhibiting a strong positive signal in the microvasculature (small arrow), intra-alveolar macrophages (large arrowheads), and granulocytes (small arrows). The positivity in bronchial epithelial cells (large arrows) is shown in the case of end-stage cystic fibrosis with acute bronchitis (C, 1:500). No positive signal was determined in the endothelial cells (small arrows) in normal lung tissue without inflammation, but a positivity in the bronchial epithelial cells (large arrows) of a bronchiole is visible (D, 1:300). Isotype controls yielded negative results in each case.

In addition to the endothelial cells, GM-CSF was expressed in bronchial epithelial cells, intra-alveolar macrophages, and granulocytes (Fig. 4).

By contrast, in uninflammed lung tissue, expression of GM-CSF in the microvasulature was absent, but bronchial epithelial cells exhibited a positive signal both in the uninflammed bronchi and in acute bronchitis (Fig. 4, C and D). No expression in lung fibroblasts was seen.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the pathogenesis of acute and chronic lung diseases, many different cell types act together (resident cells as well as cells recruited from the circulation). Recent findings suggest a pivotal role of GM-CSF in the development of various lung diseases (2, 5, 28, 40), although a picture of the pathogenetic role of this important cytokine is still far from complete. In contrast to many previous investigations, which have concentrated on inflammatory cells, especially lung macrophages as a source of this cytokine, we focused in the present paper on the lung microvascular endothelium. Because it is located in immediate anatomical and physiological relationship to intravascular, interstitial, and alveolar cells, cytokines released by dysfunctional microvascular endothelial cells could exert local effects in a paracrine fashion. This is even more feasible when taking into account that, in the dysfunctional state of the endothelium, transsudation of blood plasma components is seen. Moreover, it represents in its entirety 30-40% of the total cells in the alveolar functional unit and thereby could be a potentially important contributor to systemic production of GM-CSF.

Our results show that primary isolated human microvascular endothelial cells from the lung, simulating an inflammatory state by activation with TNF-alpha (30 or 300 U/ml), IL-1beta (100 U/ml), and LPS (0.1/1 µg/ml), are able to produce considerable amounts of GM-CSF. However, quiescent HPMEC showed neither transcriptional activity nor release of the GM-CSF protein over a period of 72 h. After activation for 24 h with LPS, a strong signal for the GM-CSF transcript was observed, demonstrating that de novo synthesis of GM-CSF is taking place. In accordance with Lenhoff and Olofsson (27), GM-CSF production in HUVEC is inducible with LPS, TNF-alpha , or IL-1beta . We confirmed the observation of Takahashi et al. (39) by RT-PCR using different PCR primers that unstimulated HUVEC do not show any transcriptional activity for GM-CSF.

Differences in the activation profiles of HUVEC and HPMEC were seen. The microvascular endothelial cells from the lung responded significantly weaker (P < 0.05 after 4 h; P < 0.025 after 24 or 72 h) to IL-1beta (100 U/ml) than did HUVEC. In addition, over a period of 72 h, no release of GM-CSF by HPMEC was observed after stimulation with IL-1beta at a 10 times lower concentration. No significant difference was observed between HUVEC and HPMEC after stimulation with LPS and TNF-alpha . Previous studies concerning the secretion of urokinase-type plasminogen activator have already indicated differences between the human lung microvascular endothelium and HUVEC (38). In the bovine lung, TNF-alpha caused different effects on microvascular vs. macrovascular endothelial cell monolayers of the lung (29). These results support the fact that endothelial cells of different topography exhibit distinct properties and that therefore human microvascular endothelial cells from the lung are required when investigating interstitial lung pathology. The various endothelial cell models are not necessarily interchangeable.

Interestingly, HAEND, an angiosarcoma cell line, exhibited in contrast to HPMEC or HUVEC a constitutive expression of GM-CSF. It seems that, during sarcoma development, the control of GM-CSF gene transcription is lost in cultured angiosarcoma cells. Nevertheless, a marked upregulation of GM-CSF expression was observed after HAEND activation with TNF-alpha , IL-1beta , and LPS.

Our in vitro findings that HPMEC have the capacity to express GM-CSF were confirmed in lung tissue by immunohistochemistry. Inflammed lung tissue from peritumoral pneumonia, candida pneumonia with sepsis, and from end-stage cystic fibrosis and uninflammed lung tissue were investigated by immunohistochemistry for the expression of GM-CSF. In areas of acute inflammation, a definitive positivity of the microvasculature for GM-CSF was observed. Additionally, intra-alveolar macrophages, granulocytes, and bronchial epithelial cells exhibited a strong positivity, with the latter cells being positive in inflammed and uninflammed areas. The potential of these cells to express GM-CSF is already known from in vitro experiments (8, 21, 39). However, expression of GM-CSF by fibroblasts could not be seen. This is in marked contrast to in vitro results, which have shown a constitutive expression of GM-CSF by lung fibroblasts (22).

We were able to demonstrate that the lung microvasculature is a potential contributor to the cytokine network in lung disease. The mechanism by which GM-CSF might initiate or accelerate pathological lung processes is poorly understood. In early phase inflammation, recruitment of inflammatory cells is of particular importance. In vitro and in vivo findings suggest that GM-CSF promotes inflammatory cell adhesion to the endothelium and could potentially assist in transmigration of inflammatory cells (4, 11, 37, 44, 49).

An increase in the number of inflammatory cells induced by GM-CSF could also be because of either an enhanced lifespan or possibly proliferative effects. HPMEC are in close vicinity both to intravascular and interstitial inflammatory cells and could, via the expression of GM-CSF, modulate the activity of inflammatory cells. In mice as well as in vitro, GM-CSF has been shown to upregulate neutrophil and eosionophil activity by increasing their survival and function (9, 12, 32, 41, 46). GM-CSF has also been described as exerting proliferative effects on alveolar macrophages, which are increased in acute and chronic lung diseases (25, 42). It would appear that GM-CSF is particularly upregulated in early stage lung diseases and that the development of a fibrotic lung reaction after overexpression of GM-CSF in the rat lung is mainly attributable to an increase in TGF-beta (2).

Besides the above-mentioned local effects, GM-CSF could exert systemic effects, e.g., on hematopoesis, because in the circulation it has a biphasic half-life (t1/2) of 10 min followed by a second t1/2 of 85 min (11).

To our knowledge, this is the first report that activated human lung microvascular endothelial cells produce GM-CSF, which has recently been shown to be pathogenetically important in various lung diseases.

In conclusion, we postulate that the microvasculature, which holds a central position anatomically and physiologically, via its production of GM-CSF, could be a crucial contributor to the pulmonary production of this important cytokine. Although much work still has to be done to elucidate the sequential pathomechanisms, our observation supports the hypothesis that the dysfunctional pulmonary microvascular endothelium, that is, activated by proinflammatory stimuli, could contribute to the induction of the interstitial fibrosis characteristic of late-stage ARDS.


    ACKNOWLEDGEMENTS

We acknowledge the excellent technical assistence of Christine Ziegelmayer, Luise Meyer, and Felicia Grimm. We also thank Dr. Gerhard Hommel (Institute for Medical Statistics, University of Mainz) for support in the statistical evaluation of the data.


    FOOTNOTES

Address for reprint requests and other correspondence: C. J. Kirkpatrick, Institute of Pathology, Johannes Gutenberg Univ., Langenbeckstrasse 1, 55101 Mainz, Germany (E-mail: kirkpatrick{at}pathologie.klinik.uni-mainz.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

March 22, 2002;10.1152/ajplung.00249.2001

Received 3 July 2001; accepted in final form 18 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abdelaziz, MM, Devalia JL, Khair OA, Calderon M, Sapsford RJ, and Davies RJ. The effect of conditioned medium from cultured human bronchial epithelial cells on eosinophil and neutrophil chemotaxis and adherence in vitro. Am J Respir Cell Mol Biol 12: 728-737, 1995.

2.   Andreutti, D, Gabbiani G, and Neuville P. Early granulocyte-macrophage colony-stimulating factor expression by alveolar inflammatory cells during bleomycin-induced rat lung fibrosis. Lab Invest 78: 1493-1502, 1998[ISI][Medline].

3.   Bayram, H, Devalia JL, Sapsford RJ, Ohtoshi T, Miyabara Y, Sagai M, and Davies RJ. The effect of diesel exhaust particles on cell function and release of inflammatory mediators from human bronchial epithelial cells in vitro. Am J Respir Cell Mol Biol 18: 441-448, 1998[Abstract/Free Full Text].

4.   Bittleman, DB, Erger RA, and Casale TB. Cytokines induce selective granulocyte chemotactic responses. Inflamm Res 45: 89-95, 1996[ISI][Medline].

5.   Bodey, KJ, Semper AE, Redington AE, Madden J, Teran LM, Holgate ST, and Frew AJ. Cytokine profiles of BAL T cells and T-cell clones obtained from human asthmatic airways after local allergen challenge. Allergy 54: 1083-1093, 1999[ISI][Medline].

6.   Brown, GM, Brown DM, Donaldson K, Drost E, and MacNee W. Neutrophil sequestration in rat lungs. Thorax 50: 661-667, 1995[Abstract].

7.   Christinsen, PJ, Balie MB, Goodman RE, O'Brien AD, Toews GB, and Paine R, III. Role of diminished epithelial GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 279: L487-L495, 2000[Abstract/Free Full Text].

8.   Cotter, TP, Hood PP, Costello JF, and Sampson AP. Exposure to systemic prednisolone for 4 h reduces ex vivo synthesis of GM-CSF by bronchoalveolar lavage cells and blood mononuclear cells of mild allergic asthmatics. Clin Exp Allergy 29: 1655-1662, 1999[ISI][Medline].

9.   Coxon, A, Tang T, and Mayadas TN. Cytokine-activated endothelial cells delay neutrophil apoptosis in vitro and in vivo. A role for granulocyte/macrophage colony-stimulating factor. J Exp Med 190: 923-34, 1999[Abstract/Free Full Text].

10.   Devalia, JL, Bayram H, Abdelaziz MM, Sapsford RJ, and Davies RJ. Differences between cytokine release from bronchial epithelial cells of asthmatic patients and nonasthmatic subjects: effect of exposure to diesel exhaust particles. Int Arch Allergy Immunol 118: 437-439, 1999[ISI][Medline].

11.   Devereux, S, and Linch DC. Granulocyte-macrophage colony-stimulating factor. In: Cytokines, edited by Mire-Sluis A, and Thorpe R.. London: Academic, 1998, p. 261-275.

12.   Fossati, G, Mazzucchelli I, Gritti D, Ricevuti G, Edwards SW, Moulding DA, and Rossi ML. In vitro effects of GM-CSF on mature peripheral blood neutrophils. Int J Mol Med 1: 943-951, 1998[ISI][Medline].

13.   Goodman, ER, Stricker P, Velavicius M, Fonseca R, Kleinstein E, Lavery R, Deitch EA, Hauser CJ, and Simms HH. Role of granulocyte-macrophage colony-stimulating factor and its receptor in the genesis of acute respiratory distress syndrome through an effect on neutrophil apoptosis. Arch Surg 134: 1049-1054, 1999[Abstract/Free Full Text].

14.   Hewett, PW, and Murray JC. Human microvessel endothelial cells: isolation, culture and characterization. In Vitro Cell Dev Biol 4: 823-830, 1993.

15.   Hoover, ML, Vetvicka V, Hoffpauir JM, and Tamburro CH. Human endothelial cell line from an angiosarcoma. In Vitro Cell Dev Biol 29A: 199-202, 1993.

16.   Huffman Reed, JA, Rice WR, Zsengeller ZK, Wert SE, Dranoff G, and Whitsett JA. GM-CSF enhances lung growth and causes alveolar type II epithelial cell hyperplasia in transgenic mice. Am J Physiol Lung Cell Mol Physiol 273: L715-L725, 1997[Abstract/Free Full Text].

17.   Jaffe, EA, Nachman NL, Becker CG, and Minick CR. Culture of human endothelial cells derived from umbilical veins. J Clin Invest 52: 2745-2756, 1973[ISI][Medline].

18.   Kantar, M, Kultursay N, Kutukculer N, Akisu M, Cetingul N, and Caglayan S. Plasma concentrations of granulocyte-macrophage colony-stimulating factor and interleukin-6 in septic and healthy preterms. Eur J Pediatr 159: 156-157, 2000[ISI][Medline].

19.   Kirkpatrick, CJ, Bittinger F, Klein CL, Hauptmann S, and Klosterhalfen B. The role of the microcirculation in multiple organ dysfunction syndrome (MODS): a review and perspective. Virchows Arch 427: 461-476, 1996[ISI][Medline].

20.   Kirkpatrick, CJ, Wagner M, Hermanns I, Klein CL, Kohler H, Otto M, van Kooten TG, and Bittinger F. Physiology and cell biology of the endothelium: a dynamic interface for cell communication. Int J Microcirc Clin Exp 17: 231-240, 1997[ISI][Medline].

21.   Klapproth, H, Racke K, and Wessler I. Acetylcholine and nicotine stimulate the release of granulocyte-macrophage colony stimulating factor from cultured human bronchial epithelial cells. Naunyn Schmiedebergs Arch Pharmacol 357: 472-475, 1998[ISI][Medline].

22.   Koyama, S, Sato E, Masubuchi T, Takamizawa A, Nomura H, Kubo K, Nagai S, and Izumi T. Human lung fibroblasts release chemokinetic activity for monocytes constitutively. Am J Physiol Lung Cell Mol Physiol 275: L223-L230, 1998[Abstract/Free Full Text].

23.   Kuhns, DB, Alvord WG, and Gallin JI. Increased circulating cytokines, cytokine antagonists, and E-selectin after intravenous administration of endotoxin in humans. J Infect Dis 171: 145-152, 1995[ISI][Medline].

24.   Kwon, EM, and Sakamoto KM. The molecular mechanism of action of granulocyte-macrophage colony-stimulating factor. J Investig Med 44: 442-446, 1996[ISI][Medline].

25.   Lehnert, BE, Valdez YE, Lehnert NM, Park MS, and Englen MD. Stimulation of rat and murine alveolar macrophage proliferation by lung fibroblasts. Am J Respir Cell Mol Biol 11: 375-385, 1994[Abstract].

26.   Lehr, HA, Bittinger F, and Kirkpatrick CJ. Microcirculatory dysfunction in sepsis: a pathogenetic basis for therapy? J Pathol 190: 373-386, 2000[ISI][Medline].

27.   Lenhoff, S, and Olofsson T. Cytokine regulation of GM-CSF and G-CSF secretion by human umbilical cord vein endothelial cells (HUVEC). Cytokine 8: 702-709, 1996[ISI][Medline].

28.   Matute-Bello, G, Liles WC, Radella F, 2nd, Steinberg KP, Ruzinski JT, Hudson LD, and Martin TR. Modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte-marcrophage colony-stimulating factor during the course of acute respiratory distress syndrome. Crit Care Med 28: 1-7, 2000[ISI][Medline].

29.   Meyrick, B, Christman B, and Jesmok G. Effects of recombinant tumor necrosis factor-alpha on cultured pulmonary artery and lung microvascular endothelial monolayers. Am J Pathol 138: 93-101, 1991[Abstract].

30.   Miyajima, A, Mui AL, Ogorochi T, and Sakamaki K. Receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5. Blood 82: 1960-1974, 1993[ISI][Medline].

31.   Moore, BB, Coffey MJ, Christensen P, Sitterding S, Ngan R, Wilke CA, Mc Donald R, Phare SM, Peters-Golden M, Paine RIII, and Toews GB. GM-CSF regulates bleomycin-induced pulmonary fibrosis via a prostaglandin-dependent mechanism. J Immunol 165: 4032-4039, 2000[Abstract/Free Full Text].

32.   Nagata, M, Sedgwick JB, Kita H, and Busse WW. Granulocyte macrophage colony-stimulating factor augments ICAM-1 and VCAM-1 activation of eosinophil function. Am J Respir Cell Mol Biol 19: 158-166, 1998[Abstract/Free Full Text].

33.   Piguet, PF, Grau GE, and de Kossodo S. Role of granulocyte-macrophage colony-stimulating factor in pulmonary fibrosis induced in mice by bleomycin. Exp Lung Res 19: 579-587, 1993[ISI][Medline].

34.   Reed, JA, and Whitsett JA. Granulocyte-macrophage colony-stimulating factor and pulmonary surfactant homeostasis. Proc Assoc Am Physicians 110: 321-332, 1998[ISI][Medline].

35.   Rimensberger, PC, Fedorko L, Cutz E, and Bohn DJ. Attenuation of ventilator-induced acute lung injury in an animal model by inhibition of neutrophil adhesion by leumedins (NPC 15669). Crit Care Med 26: 548-555, 1998[ISI][Medline].

36.   Seymour, JF, Begley CG, Dirksen U, Presneill JJ, Nicola NA, Moore PE, Schoch OD, van Asperen P, Roth B, Burdach S, and Dunn AR. Attenuated hematopoietic response to granulocyte-macrophage colony-stimulating factor in patients with acquired pulmonary alveolar proteinosis. Blood 92: 2657-2667, 1998[Abstract/Free Full Text].

37.   Smith, WB, Gamble JR, and Vadas MA. The role of granulocyte-macrophage and granulocyte colony-stimulating factors in neutrophil transendothelial migration: comparison with interleukin-8. Exp Hematol 22: 329-334, 1994[ISI][Medline].

38.   Takahashi, K, Uwabe Y, Sawasaki Y, Kiguchi T, Nakamura H, Kashiwabara K, Yagyu H, and Matsuoka T. Increased secretion of urokinase-type plasminogen activator by human lung microvascular endothelial cells. Am J Physiol Lung Cell Mol Physiol 275: L47-L54, 1998[Abstract/Free Full Text].

39.   Takahashi, M, Kitagawa S, Masuyama JI, Ikeda U, Kasahara T, Takahashi YI, Furukawa Y, Kano S, and Shimada K. Human monocyte-endothelial cell interaction induces synthesis of granulocyte-macrophage colony-stimulating factor. Circulation 93: 1185-1193, 1996[Abstract/Free Full Text].

40.   Tanaka, N, Watanabe J, Kitamura T, Yamada Y, Kanegasaki S, and Nakata K. Lungs of patients with idiopathic pulmonary alveolar proteinosis express a factor which neutralizes granulocyte-macrophage colony stimulating factor. FEBS Lett 442: 246-250, 1999[ISI][Medline].

41.   Vancheri, C, Gauldie J, Bienenstock J, Cox G, Scicchitano R, Stanisz A, and Jordana M. Human lung fibroblast-derived granulocyte-macrophage colony stimulating factor (GM-CSF) mediates eosinophil survival in-vitro. Am J Respir Cell Mol Biol 1: 289-295, 1989[ISI][Medline].

42.   Worgall, S, Singh R, Leopold PL, Kaner RJ, Hackett NR, Topf N, Moore MAS, and Crystal RG. Selective expansion of alveolar macrophages in vivo by adenovirus-mediated transfer of the murine granulocyte-macrophage colony-stimulating factor cDNA. Blood 93: 655-666, 1999[Abstract/Free Full Text].

43.   Xing, Z, Braciak T, Ohkawara Y, Sallenave JM, Foley R, Sime PJ, Jordana M, Graham FL, and Gauldie J. Gene transfer for cytokine functional studies in the lung: the multifunctional role of GM-CSF in pulmonary inflammation. J Leukoc Biol 59: 481-488, 1996[Abstract].

44.   Xing, Z, Ohkawara Y, Jordana M, Graham F, and Gauldie J. Transfer of granulocyte-macrophage colony-stimulating factor gene to rat lung induces eosinophilia, monocytosis, and fibrotic reactions. J Clin Invest 97: 1102-1110, 1996[Abstract/Free Full Text].

45.   Xing, Z, Tremblay GM, Sime PJ, and Gauldie J. Overexpression of granulocyte-macrophage colony-stimulating factor induces pulmonary granulation tissue formation and fibrosis by activation of transforming growth factor-beta 1 and myofibroblast accumulation. Am J Pathol 150: 59-66, 1997[Abstract].

46.   Yamamoto, H, Sedgwick JB, Vrtis RF, and Busse WW. The effect of transendothelial migration on eosinophil function. Am J Respir Cell Mol Biol 23: 379-388, 2000[Abstract/Free Full Text].

47.   Yasui, S, Nagai A, Aoshiba K, Ozawa Y, Kakuta Y, and Konno K. A specific neutrophil elastase inhibitor (ONO-5046. Na) attenuates LPS-induced acute lung inflammation in the hamster. Eur Respir J 8: 1293-1299, 1995[Abstract/Free Full Text].

48.   Yokoi, K, Mukaida N, Harada A, Watanabe Y, and Matsushima K. Prevention of endotoxemia-induced acute respiratory distress syndrome-like lung injury in rabbits by a monoclonal antibody to IL-8. Lab Invest 76: 375-384, 1997[ISI][Medline].

49.   Yong, KL, Rowles PM, Patterson KG, and Linch DC. Granulocyte-macrophage colony-stimulating factor induces neutrophil adhesion to pulmonary vascular endothelium in vivo: role of beta 2 integrins. Blood 80: 1565-1575, 1992[Abstract].

50.   Zsengeller, ZK, Reed JA, Bachurski CJ, LeVine AM, Forry-Schaudies S, Hirsch R, and Whitsett JA. Adenovirus-mediated granulocyte-macrophage colony-stimulating factor improves lung pathology of pulmonary alveolar proteinosis in granulocyte-macrophage colony-stimulating factor-deficient mice. Hum Gene Ther 9: 2101-2109, 1998[ISI][Medline].


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