INTRODUCTION

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Nitric oxide (NO) is a highly reactive second messenger synthesized by a group of closely related hemoproteins, nitric oxide synthases (NOS), which catalyse L-arginine conversion to NO and L-citrulline. Three main NOS isoforms have been identified so far; two of them are constitutively expressed and were first identified in the endothelial cells (endothelial isoform, ecNOS) and brain cells (neuronal isoform, nNOS) (1). The activity of these two isoforms is dependent on the presence of NADPH, thiol, L-arginine, Ca²⁺, calmodulin, and tetrahydrobiopterin (1). A third isoform, inducible NOS (iNOS), has been identified in a variety of cells in response to inflammatory cytokines and bacterial endotoxin (1). Although the requirement for NADPH and L-arginine for iNOS activity is absolute, FAD, thiol, and tetrahydrobiopterin are only partially needed, and Ca²⁺ and calmodulin are not needed. It has been established recently that numerous pulmonary cells are capable of synthesizing and releasing NO, including macrophages, neutrophils, neurons, endothelial, smooth muscle, and epithelial and mast cells (2).

Septic shock syndrome represents a major cause of death in intensive care units and is usually associated with a severe reduction in arterial pressure, high cardiac output, and intensive peripheral vasodilation (6). Bacterial lipopolysaccharide (LPS), the outer membrane of gram-negative bacteria, is known to play a central role in the pathogenesis of septic shock by activating the release of mediators and cytokines from a variety of cells (7). In the lungs, endotoxemia elicits noncardiogenic pulmonary edema and increased lung permeability (7). In humans, the presence of LPS correlates with the subsequent development of adult respiratory distress syndrome (7).

It is becoming increasingly clear that enhanced NO release (caused by direct effects of LPS or indirectly by inflammatory cytokines) plays a significant role in the pathogenesis of the vascular and pulmonary dysfunctions of septic shock. In the vascular system, hypotension and loss of vascular reactivity to vasoactive agonists in animal models of septic shock have been attributed to enhanced NO formation through activation of endothelial NOS isoform (acute phase of shock) and induction of iNOS in vascular smooth muscle cells (delayed phase of shock) (8). Numerous investigators have confirmed that NO and its derivative, peroxynitrite (formed by the reaction of NO with superoxide anions), are produced at significantly elevated concentrations in the lungs of endotoxemic animals (8, 9) and humans with ARDS (10, 11). Enhanced NO production has also been detected in the exhaled air of endotoxemic animals (12, 13) and appears to correlate with circulating NO concentration (13).

Despite the above-mentioned evidence of an important role for NO in the pathogenesis of sepsis-induced lung injury, the exact cells responsible for increased pulmonary NO production during the course of septic shock remain unclear. This is because LPS and inflammatory cytokines are capable of inducing iNOS expression and increasing the activity of constitutive NOS isoforms in numerous pulmonary cells such as alveolar macrophages and pulmonary interstitial, endothelial, and epithelial cells.

In this study, we evaluated the contribution of macrophages to enhanced pulmonary NO production in LPS-injected rats by pretreating these animals with gadolinium chloride (GdCl₃), a trivalent rare earth metal that selectively blocks the phagocytic function of macrophages (14). Our results indicate that pretreatment with GdCl₃ eliminated the rise in lung NOS activity and iNOS protein expression, and it significantly attenuated the increase in exhaled NO concentration observed after LPS injection. GdCl₃, on the other hand, had no effect on arterial pressure. These results indicate that macrophage activation plays a critical role in iNOS induction and enhanced pulmonary NO production in endotoxemia but is of less importance in LPS-induced hypotension.

	METHODS

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Reagents

Materials for L-citrulline assay were obtained from Sigma (St. Louis, MO). L-[2, 3³H]arginine was obtained from Dupont Inc. (Boston, MA). Western blotting apparatus, and gels were obtained from Novex Inc. (San Diego, CA). Monoclonal anti-iNOS antibody was obtained from Transduction Laboratories (Lexington, KY). ECL detection kit was obtained from Amersham Canada Corp. (Oakville, ON, Canada).

Animal Preparation

The procedures were approved by the Animal Research Committee of McGill University. Eighteen pathogen-free male Sprague-Dawley rats weighing 250 to 300 g were studied 1 wk after arrival. All animals were anesthetized intraperitoneally with 0.3 ml sodium pentorbarbital (40 mg/kg) and were given supplemental doses as needed. The animals were tracheostomized with polyethylene tubing (I.D., 1.68 mm; Intramedic, Clay Adams, NJ), which was sutured firmly in place with a silk tie. The lungs were ventilated mechanically with a volume-cycled ventilator (Harvard Rodent Ventilator Model 683; Harvard Apparatus, South Natick, MA), using a tidal volume of 2 to 3 ml, a respiratory rate of 40 to 60 breaths/min, and an inspired oxygen fraction of 1.0 to maintain a pH of 7.35 to 7.45, a Pa_O2 > 100 mm Hg, and a Pa_CO2 of 30 to 40 mm Hg. To ensure complete muscle paralysis, animals were injected intravenously with pancuronium bromide (0.4 mg), and additional doses were administered as needed. An arterial catheter (22-gauge) was placed into the internal carotid artery and used for measuring blood pressure (Trantec Model 60-800; American Edwards Laboratories, Santa Ana, CA) and for the sampling of arterial blood. Arterial blood gases were analyzed by means of an automatic blood gas analyzer (AVL Model 995; Instrumentation Laboratories, Lexington, MA). A catheter placed into the jugular vein was used to access the venous circulation. All animals were monitored for 5 h during the experimental protocol and then killed with an overdose of sodium pentobarbital. The chest was then opened and tissue samples were obtained from the left and right lungs and were quickly frozen in liquid nitrogen. We avoided the inclusion of the trachea and main bronchi in the lung samples. The importance of this procedure will be discussed below.

Bronchoalveolar Lavage

In 12 animals, three animals in each of the four animal groups (see EXPERIMENTAL PROTOCOL below), BAL was performed at the end of the experimental period after anesthesia with sodium pentobarbital. BAL was performed by injecting seven 5-ml aliquots of PBS (pH, 7.4) through a silastic catheter and fluid was then collected by gentle suction. BAL fluid was centrifuged for 10 min at 1,800 rpm. Cell pellet was then washed with PBS, counted in a hemocytometer, and then plated on slides (100,000 cells per slide) using a Cytospin 2 (Shandon, Runcorn, Cheshire, UK).

Immunohistochemistry

Alveolar macrophages in the BAL fluid were identified with a monoclonal ED1 antibody (Biosource International, Camarillo, CA), which reacts with all lung macrophages (15). Expression of iNOS in BAL cells was detected with a monoclonal anti-iNOS antibody (1:500), which was raised against a 21-kD protein fragment corresponding to mouse macrophage iNOS. Slides were hydrated with PBS, fixed with 4% paraformaldehyde for 10 min, and then blocked for 1 h with 10% donkey serum. After several washes, primary ED1 or iNOS antibodies were applied overnight at 4° C. Slides were then probed for 2 h at room temperature with a Cy3-labeled donkey antimouse antibody (Jackson ImmunoChemical Inc., West Grove, PA). Sites of immunoreactivity were visualized with a Nikon fluorescence microscope equipped with a Cy3 filter. Positive cells were localized and enumerated by counting a minimum of 10 random high power fields (×40 objective).

Exhaled NO Measurements

Exhaled air was collected over a 15-min period by connecting the expiratory port of the ventilator to a 5-L nondiffusing gas collection bag (Hans Rudolph, Kansas City, MO). The NO in exhaled air, thus collected, was measured by a chemiluminescence analyzer (Model 270; Sievers, Boulder, CO). Fifty-ml aliquots of the gas were introduced from the collection bag into the analyzer through a modified purging chamber, which was continuously flushed with an inert gas (nitrogen, N₂) at a flow rate of 45 to 50 ml/min. The electrical signals were amplified with a Gould Amplifier Model 6600 (Gould Instruments Division, Cleveland, OH) and were integrated to measure the area under the curve with an integration time of 2.0 s. The NO analyzer was calibrated by injecting different volumes of a certified NO gas (10 ppm balanced N₂; Medigas Inc., Holbrook, NY) with establishment of a linear calibrated scale (range, 2 to 50 pmol of NO; r² = 0.99) of NO amount (pmol) versus analyzer output (mm). The NO concentration in exhaled air was expressed as parts per billion (ppb) and calculated as the quotient of the NO content in the sample and the sample volume (50 ml).

L-Citrulline Assay

Details of the assay have been published previously (16). Frozen lung samples were homogenized in 6 volumes (wt/vol) of homogenization buffer (pH, 7.4; 10 mM HEPES buffer, 0.1 mM EDTA, 1 mM dithioreitol, 1 mg/ml PMSF, 0.32 mM sucrose, 10 µg/ml leuopeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin A). The crude homogenates were centrifuged at 4° C for 15 min at 10,000 rpm. The supernatant (50 µl) was added to 10-ml prewarmed (37° C) tubes containing 100 µl of reaction buffer of the following composition: 50 mM KH₂PO₄, 60 mM valine, 1.5 mM NADPH, 10 mM FAD, 1.2 mM MgCl₂, 2 mM CaCl₂, 1 mg/ml bovine serum albumin, 1 µg/ml calmodulin, 10 µM tetrahydrobiopterin and 25 µl of 120 µM stock L-[2, 3³H]arginine (150 to 200 cpm/pM). The samples were incubated for 30 min at 37° C, and the reaction was terminated by the addition of cold (4° C) stop buffer (pH, 5.5; 100 mM HEPES, 12 mM EDTA). To obtain free L-[³H]citrulline for the determination of enzyme activity, 2 ml of Dowex 50w resin (8% cross-linked, Na⁺ form) were added to eliminate excess L-[2, 3³H]arginine. The supernatant was assayed for L-[³H]citrulline by using liquid scintillation counting. Enzyme activity was expressed in pmol/min/mg total protein. Protein concentration was measured by the Bradford technique with bovine serum albumin as standard (BioRad Laboratories, Richmond, CA). NOS activity was also measured in the presence of 1.5 mM of each EGTA and EDTA that replaced CaCl₂ and calmodulin in the reaction buffer and in the presence of 1 mM of N^G-nitro-L-arginine methyl ester (NOS inhibitor). Ca²⁺/calmodulin-dependent NOS activity was calculated as the difference between that measured in the presence of Ca²⁺ and calmodulin and that measured in EDTA/EGTA buffer. Ca²⁺/ calmodulin-independent NOS activity was calculated as the difference between samples assayed in the presence of EGTA/EDTA and those measured in the presence of N^G-nitro-L-arginine methyl ester.

Immunoblotting

Crude homogenate proteins (80 µg) were heated for 15 min at 90° C and then loaded on gradient (4 to 12%) TRIS-Glycine SDS-polyacrylamide electrophoresis. Proteins were electrophoretically transferred onto PVDF membranes and were blocked overnight (4° C) with 5% nonfat dry milk and subsequently incubated with primary monoclonal anti-iNOS (1:500) antibody. Lysate of cytokine-activated murine macrophages was used as a positive control. Specific proteins were detected using horseradish-peroxidase-conjugated antimouse secondary antibody and chemiluminescence reagents provided with the ECL kit (Amersham Canada). The blots were scanned with an imaging densitometer (Model GS700; BioRad Inc., 12-bit precision and 42 µm resolution) and optical densities of protein bands were quantified with a software (SigmaGel; Jandel Scientific, San Rafael, CA). Predetermined MW standards (Novex Inc., San Diego, CA) were used as markers.

Experimental Protocol

Four groups of animals were studied. Group 1 (n = 6, Saline) received an injection of normal saline intraperitoneally after baseline measurements of mean arterial pressure, blood gases, and exhaled NO concentration were obtained. Group 2 (n = 6, LPS) received an intraperitoneal injection of E. coli lipopolysaccharide (LPS, serotype 055:B5, 20 mg/kg). Group 3 (n = 6, GdCl₃+Saline) animals were pretreated intravenously with 7 mg/kg gadolinium chloride dissolved in 0.1 ml sterile PBS. Twenty-four hours later, the animals received normal saline intraperitoneally as in Group 1, and the same surgical procedures and measurements of experimental variables mentioned above were performed. Group 4 (n = 6, GdCl₃+LPS) animals received gadolinium chloride intravenously 24 h prior to the experimental day. On the experimental day Group 4 animals were injected intraperitoneally with E. coli LPS (20 mg/kg), as in Group 2, and the same surgical procedures and measurements of experimental variables mentioned above were performed. Mean arterial pressure and exhaled NO concentration in all animals were measured every 30 min during the experimental period (5 h).

Statistical Analysis

All values are expressed as mean ± standard error of the means (SEM), except for BAL cytology. Statistical analysis was performed with one-way analysis of variance (ANOVA) with Scheffe's test for multiple comparison. Student's t test was used for comparison between two groups. A p value < 0.05 was regarded as statistically significant difference.

	RESULTS

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The changes in arterial pressure in the four groups of animals are illustrated in Figure 1. Although mean arterial pressure remained unchanged in the Saline and GdCl₃+Saline groups, it declined progressively in the LPS group. Pretreatment with GdCl₃ had no effect on LPS-induced hypotension (Figure 1).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Changes in mean arterial pressure in the four groups of animals. Notice that arterial pressure in the Saline and GdCl3+Saline remained unchanged, whereas it declined progressively in the LPS and GdCl3+LPS groups.

The changes in arterial blood gases before (baseline) and after 300 min of experimental period in the four groups of animals are listed in Table 1. There was a significant decline in arterial pH in the LPS and GdCl₃+LPS groups, whereas pH remained unchanged in the other two groups.

The changes in exhaled NO concentration in the four groups are shown in Figure 2. Exhaled NO concentration remained unchanged in the Saline and GdCl₃+Saline groups from a baseline value of about 7 ppb (Figure 2). LPS injection elicited a more than 4-fold rise in exhaled NO concentration, which reached a plateau after 200 min of LPS injection. Pretreatment with GdCl₃ resulted in a smaller rise (2-fold) in exhaled NO concentration compared with the LPS group (Figure 2). However, exhaled NO concentration measured at 120 min onward in the GdCl₃+LPS group was significantly higher than that of the Saline and GdCl₃+Saline groups (Figure 2).

View larger version (21K): [in this window] [in a new window]   Figure 2.   The time course of exhaled NO concentration in the four groups of animals. Exhaled NO concentration remained similar to baseline values (0 time) throughout the experimental period in the Saline and GdCl3+Saline group. In the LPS group, exhaled NO concentration was significantly higher than the baseline value after 120 min of LPS injection. Pretreatment with GdCl3 attenuated the rise in exhaled NO (p < 0.01 compared with LPS group at 150 min onward). However, exhaled NO concentration in the LPS and GdCl3+LPS groups was significantly higher than the saline group at 120 min onward.

Injection of LPS elicited a substantial rise in lung Ca²⁺/ calmodulin-dependent and independent NOS activities (p < 0.01 compared with the Saline and GdCl₃+Saline groups (Figure 3). Pretreatment with GdCl₃ eliminated LPS-induced augmentation of pulmonary NOS activity (Figure 3).

View larger version (16K): [in this window] [in a new window]   Figure 3.   Total and Ca2+/calmodulin-independent lung nitric oxide synthase activities in the four groups of animals. **p < 0.01 compared with the Saline group. Notice that GdCl3 pretreatment completely abrogated the rise in lung NOS activity observed after LPS injection.

An immunoblot of lung samples obtained after 5 h of experimental period in the four groups of animals is shown in Figure 4. We detected a weak 130 kD iNOS protein band in the lungs of the saline-injected rats. Injection of LPS elicited a significant upregulation of iNOS protein expression (OD of 200% of the saline group). Pretreatment with GdCl₃ completely eliminated this rise in iNOS protein expression observed after LPS injection (OD of only 3% of the saline group) (Figure 4). The intensity of iNOS protein band in the lungs of GdCl₃+ Saline group was slightly weaker than that of the saline group (declined OD of 60% of the saline group).

View larger version (24K): [in this window] [in a new window]   Figure 4.   A representative immunoblotting of lung tissue samples with monoclonal anti-iNOS antibody. Equal amounts of lung homogenates (80 µg) were loaded per lane. Notice that weak iNOS expression was detected in the Saline and GdCl3+Saline group, whereas strong iNOS expression was detected in the LPS group. GdCl3 pretreatment eliminated iNOS expression in the GdCl3+LPS group.

To confirm whether or not GdCl₃ pretreatment influenced pulmonary macrophage NO production, we examined BAL cells for iNOS protein expression. We applied immunostaining with ED1 antibody, which reacts with all pulmonary macrophages (15) to identify these cells from other pulmonary cells normally present in BAL fluid. Our results indicate that in the four groups of rats, more than 90% of BAL cells were macrophages. The iNOS protein expression by lung macrophages in various animal groups is illustrated in Figure 5. In saline-injected animals, the number of BAL cells stained positive for iNOS averaged 8 ± 4 (mean ± SD) of total cells (Figure 5A). Pretreatment of Saline-injected animals with GdCl₃ did not alter the percentage of iNOS-positive BAL cells (10 ± 3%) from that observed in the saline group. In LPS-injected rats, 65 ± 12% of BAL cells stained positive for iNOS (Figure 5B). Positive staining was clearly limited to the cytoplasm, with no evidence of iNOS staining in the nuclei. Double immunostaining for ED1 and iNOS in one animal indicated that iNOS expression was limited to the macrophages. Preteatment of LPS-injected rats with GdCl₃ reduced the percentage of iNOS-positive BAL cells to about 17 ± 5% of the total cells (Figure 5C). These results confirm the inhibitory influence of GdCl₃ pretreatment on the ability of pulmonary macrophages to express iNOS protein in response to in vivo endotoxemia.

View larger version (81K): [in this window] [in a new window]   Figure 5.   Expression of iNOS protein in BAL cells of various animals groups. BAL cells were immunostained for iNOS expression using primary anti-iNOS antibody and Cy-3-labeled secondary antibody. Specific iNOS staining (white color, arrows) was detected in the macrophages of LPS-injected animals (panel B), whereas very few cells were stained positive for iNOS in saline-injected animals (panel A). Pretreatment of LPS-injected animals with GdCl3 reduced the number of iNOS-positive macrophages (panel C ). Panel D represents negative control staining.

	DISCUSSION

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The main findings of this study are: (1) injection of E. coli LPS elicited a progressive decline in arterial pressure, increased pulmonary NOS activity and exhaled NO concentration, and upregulated pulmonary iNOS protein expression; (2) pretreatment with GdCl₃ eliminated the rise in pulmonary NOS activity and iNOS expression, attenuated the rise in exhaled NO concentration, but had no effect on LPS-induced hypotension. In addition, fewer pulmonary macrophages expressed iNOS protein in LPS-injected animals when these animals were pretreated with GdCl₃.

Consideration of Methods

In this study we used a selective inhibitor of macrophage phagocytic activity, GdCl₃ (17), a rare earth metal of the lanthanide series, in order to evaluate the role of macrophages in augmenting NO release in the vascular and pulmonary systems of endotoxemic rats. Pretreatment with GdCl₃ is known to prevent LPS-stimulated iNOS induction in the hepatocytes (18) and Kupffer cells (19). Little is known about the effects of GdCl₃ on pulmonary macrophage function. Bannenberg and colleagues (20) reported that phagocytic activity of pulmonary macrophages was not influenced by pretreatment with GdCl₃. These results contrast with those of Pendino and colleagues (21) who described that an injection of GdCl₃ (7 mg/kg) 48 h before ozone exposure significantly attenuated ozone-induced lung injury and pulmonary nitrite production. These investigators also found that GdCl₃ pretreatment eliminated in vitro LPS-induced iNOS expression in lung lavage cells. To confirm that GdCl₃ influenced in vivo iNOS expression in pulmonary macrophages, we examined BAL fluid for iNOS expression in the four groups of animals. Our results indicate for the first time that GdCl₃ pretreatment significantly attenuates the number of pulmonary macrophages that express iNOS protein after LPS injection. It should be noted that we detected iNOS expression in pulmonary macrophages of saline-injected animals (Figure 5). This finding, which is similar to that reported recently by Liu and colleagues (15), is suggestive of a "constitutive" iNOS expression in these cells. The functional significance of this expression to normal lung functions remains unclear.

Pulmonary NO Production

Many studies have documented that nNOS and ecNOS isoforms are expressed under normal conditions in a variety of pulmonary cells, including nerve fibers, endothelial cells, airway epithelial cells, and cultured epithelial cell lines (2, 3, 22). More recently, constitutive iNOS expression has been confirmed in large airway and tracheal epithelial cells in normal humans and macrophages of normal rats (4, 15). We detected low NOS activity and weak iNOS expression in the lungs of Saline-injected rats. This iNOS expression could be attributed to the expression of iNOS in the epithelial cells of trachea and large airways as well as in alveolar macrophages as detected with iNOS immunostaining of BAL cells (Figure 5).

NO is actively released into the exhaled air of normal humans and animals (23). The exact source of exhaled NO is still being investigated, but evidence of a large nasal epithelial contribution is accumulating (24). Our results confirm the presence of low basal NO release in the exhaled air of mechanically ventilated rats (Figure 2). We should emphasize, however, that any contribution of upper airways to exhaled NO production was excluded in our study because all of our animals were ventilated through tracheal cannulae (see METHODS).

Pulmonary NO Production in Endotoxemia

It is well known that injection of bacterial endotoxin in experimental animals elicits a widespread iNOS expression, including strong pulmonary expression (8). Recent studies have indicated that elevated NO concentration in the exhaled air is an early marker of lung injury in various animal models of septic shock (12, 13, 25). Our current results confirm that endotoxemia in rats is associated with significant elevation of lung NOS activity and iNOS induction. We have also observed a gradual rise in exhaled NO concentration in the LPS group, which is very consistent with the results of Stewart and colleagues (12). The exact cellular source of enhanced pulmonary NO production in septic shock and endotoxemia remains unclear. Although the possibility that NO could be produced by iNOS protein located in the pulmonary endothelial cells, vascular and airway smooth muscles, and alveolar type II pneumocytes, there is strong evidence supporting a major role for macrophages. Indeed, injection of LPS in rats is known to be associated with strong iNOS expression in macrophages distributed abundantly in the heart, lung, liver, and kidney (3, 26, 27). Despite this evidence of macrophage involvement, there is no information regarding the degree to which macrophages contribute to total lung NOS activity in septic shock. Our study indicates for the first time that pretreatment with the inhibitor of macrophage activation, GdCl₃, eliminates the rise in pulmonary NOS activity and iNOS expression associated with endotoxemia, suggesting a critical role for activated macrophages in the pulmonary response to LPS. We also found that GdCl₃ pretreatment attenuated, but not completely abrogated, the rise in exhaled NO concentration during the course of endotoxemia. These findings indicate that the majority of exhaled NO production in the septic animals is dependent on macrophage activation.

There are two possible pathways through which GdCl₃ pretreatment could have eliminated the significant rise in pulmonary NOS activity and iNOS expression in septic animals. Firstly, it is possible that GdCl₃ inhibited LPS-induced cytokine production by macrophages that are resident in the liver (Kupffer cells). Cytokines such as TNF and IL-1 are critical for in vivo induction of iNOS expression in various organs, including the lungs (28). Several investigators have reported that pretreatment with GdCl₃ was, indeed, associated with reduction in plasma TNF- levels in endotoxemic rats (29). Thus, one could conclude that elimination of pulmonary NOS activity and iNOS induction in the GdCl₃-LPS group was secondary to diminished cytokine production by resident macrophages in various organs. Although feasible, we believe that this is not the major mechanism explaining our results because reduction of cytokine production systemically by GdCl₃ pretreatment would have resulted in improvement of hemodynamic as well as pulmonary derangements elicited by LPS injection. The fact that the reduction of arterial pressure brought about by LPS injection was not influenced by GdCl₃ pretreatment suggests systemic cytokine production might not have been attenuated by GdCl₃. Secondly, we therefore propose that GdCl₃ directly inhibits iNOS induction in pulmonary macrophages as indicated by immunostaining of BAL macrophages (Figure 5). This proposal is further supported by the observations that in vivo pretreatment with GdCl₃ inhibits ex vivo iNOS induction in response to LPS or ozone exposure in Kupffer cells and pulmonary macrophages (19, 21). The mechanisms through which GdCl₃ interferes with iNOS induction in macrophages is not yet known. One possible mechanism is inhibition of calcium signaling. Calcium and protein kinase C are required for iNOS induction in macrophages (30), and GdCl₃ is known to displace Ca²⁺ from cation-binding sites (17).

Implication to Human Septic Shock

Although our results convincingly indicate that pulmonary macrophages express the iNOS isoform in response to LPS injection and contribute significantly to the rise in pulmonary NO production in endotoxemic animals, the involvement of these cells in regulating pulmonary NO production in septic humans remains under investigation. There is also a controversy as to whether human pulmonary macrophages are capable of producing high levels of NO and express iNOS protein. Supporting iNOS expression in human macrophages is the observation of Kobzik and colleagues (3) who detected iNOS staining in pulmonary macrophages in normal human lung samples. Others (31) confirmed a high rate of NO production by human monocytes in response to LPS. On the other hand, Weinberg and colleagues (32) proposed that human monocytes produced little NO in response to in vitro cytokines and LPS exposure. This notion is supported by the observation that U937 cells (human monocytelike cell line) are incapable of iNOS induction when compared with murine macrophages (RAW 264.7) (33). This observation was attributed to failure of iNOS transcription as a result of irregular nucleotide sequences in the enhancer element (Region II) of human iNOS promoter that are necessary for LPS/IFN--induced iNOS transcription. It is clear that more research is needed to elucidate the influence of cytokines and LPS on in vivo expression of iNOS in human macrophages.