Introduction

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The renin-angiotensin system is a bioenzymatic cascade in which renin acts on angiotensinogen to form angiotensin I, which is then converted by ACE to AII. AII is an important molecule controlling blood pressure and volume in the cardiovascular system. Its importance is manifested by the efficacy of ACE inhibitors in the treatment of hypertension and congestive heart failure (Peach, 1977; Vidt et al., 1982; Smith et al., 1992). ACE inhibitors are widely used in therapy, and many nonpeptide AII receptor antagonists are in various stages of clinical development, with losartan being the first such drug available for clinical use since 1990 (Testa et al., 1993; Keilani et al., 1995; Johnston, 1995; Clauser et al., 1996). Losartan, moreover, has become the prototypical tool used to determine the role of AII in biological systems and is the reference standard for AT1 receptors (Timmermans et al., 1993).

Previous work has shown that mononuclear phagocytes synthesize angiotensinogen, angiotensin I and AII and express receptors with high affinity for AII (Thomas and Hoffman, 1984; Gomez et al., 1993; Kitazono et al., 1995; Suzuki et al., 1995). AII appears to be able to modulate mononuclear phagocyte functions. In this regard, Dezsö and Fóris (1981) and Fóris et al. (1983) showed that AII regulates the activity of receptors for the Fc fragment of IgG, whereas Hahn et al. (1994) found that AII stimulates tumor necrosis factor- production.

Leukocytes migrate from the blood to sites of inflammation in response to locally produced chemoattractants that activate specific cell surface receptors. All of these receptors, including the receptors for bacterial N-formyl peptides, belong to the class of G-protein-coupled seven-transmembrane domain receptors (Murphy, 1994). AT1 receptors for AII also belong to this family of receptors (Timmermans et al., 1993; Clauser et al., 1996). Moreover, a database search revealed that the AT1 receptor for AII and the high affinity receptor for fMLP share 25 to 30% sequence identity (Bernstein and Alexander, 1992).

Considering the stimulatory activities of AII on mononuclear phagocyte responses and the relationship between the AT1 receptor for AII and the high-affinity receptor for fMLP, we chose to initiate research to define whether AII may modulate neutrophil activation triggered by the chemoattractant peptide fMLP. Unexpectedly, during the course of these experiments, we observed that losartan selectively inhibits neutrophil activation induced by fMLP, through a mechanism not related to the ability of losartan to antagonize angiotensin receptors.

	Materials and Methods

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Reagents. fMLP, AII, zymosan, Con A and saralasin were purchased from Sigma Chemical Co. (St. Louis, MO). Captopril was obtained from Squibb Laboratory (Paris, France); losartan and [³H]fMLP were from DuPont (Boston, MA). Precipitating IC were prepared as previously described, using human IgG as antigen and rabbit IgG antibodies to human IgG (Schattner et al., 1993).

Preparation of neutrophils and monocytes. Blood samples were obtained from healthy donors who had taken no medication for at least 10 days before the day of sampling. Blood was obtained by venipuncture of the forearm vein, and it was drawn directly into plastic tubes containing 3.8% sodium citrate (1:9, v/v). Human neutrophils were isolated by dextran sedimentation and Ficoll-Hypaque gradient centrifugation (Ficoll; Pharmacia, Uppsala, Sweden; Hypake; Winthrop Products Inc., Buenos Aires, Argentina), as described (Boyum, 1968). Contaminating erythrocytes were removed by hypotonic lysis. After washing, the cells (>96% neutrophils in May Grunwald/giemsa-stained cytopreparations) were resuspended at the desired concentration in RPMI 1640 medium (GIBCO, Detroit, MI). Peripheral blood mononuclear cells were harvested from the interphase of the Ficoll-Hypaque gradient. After washing, the cells were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated FCS (GIBCO), to a final concentration of 5 × 10⁶ peripheral blood mononuclear cells/ml. Purified cells, containing 95 to 98% mononuclear cells and 2 to 5% neutrophils, were placed in plastic Petri dishes that had been pretreated with autologous serum. After incubation for 2 hr at 37°C, nonadherent cells were removed by washing and adherent cells were detached with a rubber policeman and resuspended at the desired concentration in RPMI 1640 medium. These cell suspensions contained 75 to 90% monocytes.

CL assays. Neutrophils and monocytes were suspended at 2.5 × 10⁶/ml in culture medium supplemented with 1% FCS. Luminescence responses were measured with a Lumi-aggregometer (Chrono-Log Corp., Haverton, PA) at 1000 revolutions/min and 37°C, in the presence of luminol (0.1 µM), as previously described (Geffner et al., 1993). In all cases, light emission was continuously registered for 10 min. Data are expressed as the maximum response observed during this period, in relative CL units. One CL unit was defined as 1-cm shifting of the light emission signal on the paper recorder.

Neutrophil adherence. Adherence was assessed as previously described (Geffner et al., 1991). Briefly, neutrophils were suspended in RPMI 1640 medium supplemented with 1% FCS and were labeled with Na₂CrO₄ (1 µCi/10⁶ cells) for 1 hr at 37°C. The cells were then washed four times with saline and resuspended in RPMI 1640 medium supplemented with 10% FCS, to a density of 4 × 10⁶ cells/ml. One hundred microliters of this suspension were added to each well in 96-well, flat-bottomed, polystyrene plates. Neutrophils were incubated in the presence or absence of different compounds for 30 min at 37°C in 5% CO₂/95% humidified air and were washed three times with culture medium to remove nonadherent neutrophils. Adherent neutrophils were then lysed with 1 N NH₄OH, and the radioactivity present in the lysates was measured. Cell adherence was expressed as the number of neutrophils that remained adherent to the plastic surface after washing.

Shape change assay. This assay was performed as described previously (Craig Stocks et al., 1995). Briefly, neutrophils (1.5 × 10⁶) suspended in 100 µl of culture medium supplemented with 1% FCS were incubated in the presence or absence of different compounds, in a shaking water bath, at 37°C for 15 min. After washing, cells were suspended in phosphate-buffered saline and fixed by the addition of an equal volume of 0.5% glutaraldehyde in phosphate-buffered saline. Shape change was assayed in a fluorescence-activated cell-sorting analyzer (Becton Dickinson Immunocytometry System, San Jose, CA). Results were expressed in mean forward light scatter units.

Neutrophil fMLP receptor binding assay. Binding of fMLP to neutrophil receptors was measured, as described previously (Weisbart et al., 1986), using [³H]fMLP (specific activity, 61.5 Ci/mmol). Increasing concentrations of labeled peptide (2.5-80 nM final concentration) were added to unstimulated neutrophils in the absence or presence of losartan (5 µg/ml). After 5 min at 22°C, radiolabeled fMLP and neutrophils (3 × 10⁶/tube, in a total volume of 250 µl) were incubated on a shaking platform for 30 min in an ice bath. Nonspecific binding of [³H]fMLP to neutrophils was measured by mixing the radiolabeled ligand with 10 µM unlabeled peptide and incubating the mixture for 30 min in an ice bath. Nonspecific binding was similar in the absence and presence of losartan (5 µg/ml) and accounted for 11% of the binding. The values for nonspecific binding were subtracted from the corresponding values for total bound [³H]fMLP to yield the specifically bound radioligand. The number of binding sites and the dissociation constant were determined from a Scatchard plot analysis, using the computer program LIGAND (Munson and Rodbard, 1980).

Statistical analyses. Student's paired t test was used to determine the significance of differences between means, and P < .05 was taken as statistically significant.

	Results

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Effect of losartan on neutrophil responses triggered by different stimuli. The effect of losartan on neutrophil activation was examined by studying the following responses: CL emission, adherence and shape change. As shown in figure 1, responses induced by IC, zymosan and Con A were not affected by losartan, whereas responses induced by fMLP were, in all cases, dramatically suppressed. A dose-response experiment is shown in figure 2. Doses of 5 µg/ml significantly (P < .01) inhibited CL and adherence responses triggered by 10 nM fMLP. The ability of losartan to inhibit fMLP-triggered responses was shown to be dependent on the concentrations of both losartan and fMLP. In fact, when CL responses induced by high concentrations of fMLP (0.1 µM) were analyzed, it was observed that 10 µg/ml losartan was unable to exert any effect (data not shown). In contrast, with low concentrations of fMLP (1-3 nM), a significant inhibition of CL (inhibition, 46 ± 9%; n = 6, P < .01) was observed at doses of losartan as low as 0.5 µg/ml.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Effect of losartan on neutrophil responses triggered by different stimuli. CL emission (A), adherence (B) and shape change (C) were assessed as described in "Materials and Methods," in the absence () or presence () of losartan (10 µg/ml), which was added 5 min before the addition of stimuli. Results are expressed in relative CL units (RCLU), number of adherent cells and mean forward light scatter units (FSC), respectively. The following stimuli were used: fMLP (10 nM), IC (50 µg/ml), zymosan (50 µg/ml) (Zy) and Con A (20 µg/ml). Data are expressed as the mean ± S.E.M. of 7 to 11 experiments performed in duplicate. *P < .005 vs. neutrophils stimulated by fMLP in the absence of losartan.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Inhibition of fMLP-triggered neutrophil activation by different concentrations of losartan. CL emission () and adherence () were triggered by fMLP (10 nM). Assays were performed as described in "Materials and Methods." Percentages of inhibition were calculated at each point by comparing the responses observed in the absence and presence of losartan. Data are expressed as the mean ± S.E.M. of seven experiments performed in duplicate.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Effect of saralasin, captopril and AII on neutrophil CL responses triggered by fMLP. A, CL responses measured in the absence () or presence of the renin-angiotensin system antagonists saralasin () and captopril () (5 µg/ml); B, responses measured in the absence () or presence of 1 µg/ml () and 0.01 µg/ml () AII. In all cases the drugs were added to neutrophils 5 min before the addition of fMLP. Data are expressed in relative CL units and represent the mean ± S.E.M. of five experiments performed in duplicate.

Effect of losartan on CL emission mediated by monocytes and triggered by different stimuli. In another set of experiments, we examined the effect of losartan on monocyte activation by studying CL emission triggered by different stimuli. As observed for neutrophils, it was found that CL responses induced by IC, zymosan and Con A were not modified by losartan, whereas responses triggered by fMLP were markedly inhibited (fig. 4).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Effect of losartan on CL emission mediated by monocytes triggered by different stimuli. CL assays were performed as described in "Materials and Methods," in the absence () or presence () of losartan (5 µg/ml), which was added 5 min before the addition of stimuli. Results are expressed in relative CL units (RCLU). The following stimuli were used: fMLP (10 nM), IC (100 µg/ml), zymosan (50 µg/ml) (Zy) and Con A (20 µg/ml). Data are expressed as the mean ± S.E.M. of six to eight experiments performed in duplicate. *P < .01 vs. monocytes stimulated by fMLP in the absence of losartan.

Effect of losartan on fMLP binding to neutrophil receptors. To examine the mechanisms by which losartan suppresses neutrophil activation by fMLP, its ability to inhibit [³H]fMLP binding to neutrophils was measured by adding increasing amounts of tritiated fMLP to fractions of 5 × 10⁶ neutrophils. Specific binding was determined by adding [³H]fMLP to neutrophils in the absence and presence of 10 µMunlabeled fMLP. Scatchard analysis of fMLP binding in the presence of losartan (5 µg/ml) showed that this compound did not modify the number of binding sites but markedly decreased the binding affinity of fMLP for neutrophil receptors, with a change in K_d from 36 nM to 88 nM (fig. 5).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Effect of losartan on the binding of fMLP to neutrophils. A representative Scatchard plot for [3H]fMLP binding to neutrophils in the absence () and presence () of losartan (5 µg/ml) is shown. The assay was performed as described in "Materials and Methods." Curve-fitting was done with the LIGAND program. Each point represents the mean of duplicate determinations. The dissociation constant (Kd) of fMLP was significantly different (P < .01) in the absence (36 nM) and presence (88 nM) of losartan.

	Discussion

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In this work we demonstrate that losartan, a selective antagonist of AT1 receptors for AII, markedly suppresses the activation of neutrophils by fMLP. This effect cannot be ascribed to the ability of losartan to antagonize angiotensin receptors, because 1) neither saralasin, an inhibitor of AT1 and AT2 receptors for AII, nor captopril, an inhibitor of ACE, reproduces the effect mediated by losartan and 2) neutrophil responses triggered by fMLP are not affected by exogenously added AII. Impairment of fMLP-induced activation by losartan is due, at least in part, to its ability to inhibit neutrophil binding of fMLP. Scatchard analysis showed that losartan markedly decreased the affinity of fMLP for neutrophil receptors without affecting the number of binding sites. It is noteworthy that not only neutrophils but also monocytes were affected by losartan. In fact, we observed that losartan markedly inhibited CL emission by monocytes stimulated with fMLP.

Losartan and other AII AT1 receptor antagonists being developed for clinical use are phenyltetrazole-substituted imidazoles. Losartan potassium is a low molecular weight (molecular weight, 461) nonpeptide (hence, orally active) that shows high selectivity for the AT1 receptor subtype (Smith et al., 1992; Timmermans et al., 1993). At concentrations of 10 µM, it does not show affinity for other hormonal receptors, such as Ca⁺⁺ channels and alpha and beta adrenergic, neurotensin, glycine, opioid (µ,  and ), muscarinic, dopaminergic and serotonergic receptors (Smith et al., 1992; Timmermans et al., 1993). These data indicate that losartan is indeed a specific agent for AT1 receptors. We show, for the first time, that losartan can effectively inhibit cellular specific binding of a peptide different from AII.

After oral administration of clinically effective doses of losartan (50-100 mg), the blood concentration peak at 1 hr reaches values of 0.5 to 1.1 µg/ml (Smith et al., 1992; Timmermans et al., 1993). Our results showed that, in vitro, these concentrations of losartan are able to inhibit neutrophil responses triggered by low concentrations of fMLP. Considering the critical role that N-formyl peptides play in the recruitment and activation of phagocytic cells in response to microbial injury (Marasco et al., 1984; Murphy, 1994), it is tempting to speculate that, during therapy with losartan, there may be the risk of bacterial infectious diseases. However, observations from controlled clinical trials do not support this presumption, because losartan is well tolerated and the incidence of infectious diseases appears to be comparable to that in the placebo group (Goodfriend et al., 1996). It should be emphasized, however, that so far no clinical trials have involved immunocompromised patients, for whom the impairment of phagocytic cell ability to respond to N-formyl peptides would be more harmful, compared with immunocompetent patients. Additional studies will be required to determine whether losartan should be given to immunocompromised patients.