Pasteurella haemolytica A1-Derived Leukotoxin and Endotoxin Induce Intracellular Calcium Elevation in Bovine Alveolar Macrophages by Different Signaling Pathways

Abstract

Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381–388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237–252, 1995). Furthermore, extracellular Ca²⁺ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca²⁺ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca²⁺ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca²⁺ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca²⁺ in a concentration-dependent fashion by influx of extracellular Ca²⁺ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca²⁺ even in the absence of extracellular Ca²⁺. Leukotoxin-induced intracellular Ca²⁺ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A₂ and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca²⁺ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca²⁺ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A₂ and C and release of arachidonic acid in bovine alveolar macrophages. Ca²⁺ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca²⁺ signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.

Pasteurella haemolytica serotype 1 is the bacterial agent that contributes to peracute lung injury in bovine pneumonic pasteurellosis, a disease of considerable economic importance to the beef and dairy industries (7, 39). Leukotoxin (Lkt), which is a ∼104-kDa pore-forming RTX toxin (named RTX for repeats in toxin), secreted by this organism is considered to be the major virulence factor contributing to lung injury in the disease (38). Endotoxin (lipopolysaccharide [LPS]) derived from this organism has also been implicated in the pathogenesis of lung injury associated with the disease (37, 42, 44). In pneumonic pasteurellosis, the alveolar macrophages play a central role in orchestrating the cellular events and the inflammatory cascade leading to lung damage (38, 42). Both Lkt and LPS are known to induce the expression of genes for the proinflammatory cytokines, including interleukin 1β and tumor necrosis factor alpha in bovine alveolar macrophages (BAMs) (42, 43). Although similar profiles of proinflammatory cytokine genes are expressed in response to Lkt and LPS, they show marked differences in the kinetics of expression, and different signal transduction mechanisms may account for these differences.

A previous study has shown that Lkt stimulation of bovine neutrophils results in elevation of intracellular Ca²⁺ ([Ca²⁺]_i) by influx of extracellular Ca²⁺ through voltage-gated channels (22). Similar findings have been reported in human neutrophils by Lkt from Actinobacillus actinomycetemcomitans (12). Although these studies indicate that [Ca²⁺]_i response to Lkt may be an early event during activation of leukocytes, the precise signaling pathways leading to the [Ca²⁺]_i response are not clearly understood.

In macrophages from several species, LPS has been shown to stimulate phospholipase C (PLC) and phospholipase D, resulting in the production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) (24, 28, 29). It has been well established that IP₃ stimulates the release of Ca²⁺ from intracellular stores in many different cell types (25, 27). However, there is conflicting information on the roles of IP₃ and DAG in mobilization of intracellular Ca²⁺ by LPS in macrophages (5, 28). The results of a previous study have also indicated the role of protein tyrosine phosphorylation in LPS-induced arachidonic acid release in a murine macrophage cell line (35). In the present study, we characterized the signaling mechanisms responsible for Lkt- and LPS-induced elevation of [Ca²⁺]_i in BAMs. Our results not only demonstrate differences in signaling pathways but also provide the first direct evidence for Lkt-induced Ca²⁺ influx in BAMs through G-protein-coupled activation of phospholipase A₂ (PLA₂) and PLC.

MATERIALS AND METHODS

Preparation of P. haemolytica Lkt.

The preparation of Lkt derived from P. haemolytica D153 has been described in a previous publication (18). Briefly, P. haemolytica D153 was cultured in RPMI 1640 medium supplemented with 2 mM l-glutamine. The logarithmic-growth-phase bacterial culture supernatant was collected by centrifugation, filter sterilized, concentrated, and dialyzed against endotoxin-free distilled water in a spiral-wound membrane cartridge (model S1Y30; Amicon Corp., Danvers, Mass.). The resulting crude Lkt fraction was lyophilized and stored at −20°C. The crude Lkt was purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and purity was confirmed by the method of Yoo et al. (43). The purified Lkt was lyophilized and stored at −20°C until use, and all experiments were done with the same batch of purified Lkt. The bioactivity of Lkt was determined by the colorimetric XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) assay, using BL-3 bovine lymphoma cell line as target cells (26), and the concentration of Lkt was expressed as Lkt units per ml (43). The purified Lkt was tested for the presence of LPS contamination by using a chromogenic Limulus amebocyte lysate assay kit (BioWhittaker, Walkersville, Md.), and the level of LPS was found to be 0.0017 ng/ml.

Preparation of P. haemolytica LPS.

The P. haemolytica LPS was extracted by the hot-phenol-water method, as described by Westphal and Jann (36). LPS extract was lyophilized and stored at 4°C. The chromogenic Limulus amebocyte lysate assay (BioWhittaker) was used to quantify the bioactivity of LPS. One microgram of purified LPS was equivalent to ∼208.6 endotoxin units.

Isolation of BAMs and culture.

BAMs were isolated from five healthy 6- to 8-week-old calves by the method of Yoo et al. (43). The macrophages were frozen in Dulbecco’s modified Eagle medium (DMEM) containing 20% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen until use. Frozen cells were rapidly thawed, resuspended in DMEM supplemented with 5% fetal bovine serum, 2 mM l-glutamine, 20 mM HEPES, 100 U of penicillin per ml, 100 μg of streptomycin per ml, and 25 μg of amphotericin B per ml. The cells were plated onto round 15-mm-diameter glass coverslips at a density of 7.5 × 10⁵ cells/ml in 12-well tissue culture plates and incubated at 37°C in a humidified atmosphere containing 5% CO₂. The medium was changed every other day, and the cells were used after 4 days. The viability of the BAMs, as determined by trypan blue dye exclusion, was >98%.

Video fluorescence imaging.

The glass coverslips with attached BAMs were washed with Hank’s balanced salt solution (HBSS) containing 10 mM glucose and buffered with 10 mM HEPES (pH 7.4). The cells were incubated in 10 μM fura-2-acetoxymethyl ester (fura-2/AM) (Molecular Probes, Eugene, Oreg.) for 45 min at 37°C and then rinsed in fresh HBSS. The coverslips were mounted on a Plexiglas chamber (150-μl volume; Warner Instruments, Hamden, Conn.) and transferred to the stage of a Nikon Diaphot inverted microscope with an epifluorescence attachment. The chamber was washed with HBSS at room temperature at 2 to 3 ml/min.

The cells were visualized by using a Nikon UV-Fluor 40× oil immersion objective lens. The fluorescence excitation, image acquisition, and real-time data analyses were controlled by a dedicated video fluorescence imaging system (Image-1; Universal Imaging) running on a Pentium P5-90 personal computer. Cells loaded with fura-2/AM were alternately excited at 340 and 380 nm by a rapidly rotating filter wheel with a time delay of ∼50 ms between the excitation wavelengths. The fluorescence emissions were collected for each wavelength with a 510-nm-wavelength barrier filter. A complete set of images was acquired once every second using a silicon-intensified target video camera (MTI Corporation). No background subtraction was employed. The gain and sensitivity of the camera were set by using the basal level of fura-2 emissions as an index of cell loading. The camera settings ensured that, even with a large shift in fluorescence emissions during the actual experiment, the camera was operating within its dynamic range. In experiments involving extended periods of data collection, UV excitation was interrupted when not needed, in order to minimize dye bleaching.

The ratio of fura-2 emissions at 340 and 380 nm (F₃₄₀/F₃₈₀) was used as an index of [Ca²⁺]_i, as described in an earlier publication (15). Since the actual amplitude of the [Ca²⁺]_i response varied for different cells and protocols, the integrated [Ca²⁺]_i response, which reflects the total Ca²⁺ released during the time period of interest, was used instead for comparison of effects. The F₃₄₀/F₃₈₀ ratio is depicted in the representative traces. A preliminary image of F₃₄₀/F₃₈₀ was obtained before the experiment. Regions of interest covering ∼100 pixels were outlined using an interactive cursor. Caution was used to ensure that these regions were completely within the boundaries of a cell.

Experimental protocols.

In preliminary experiments, elevation of [Ca²⁺]_i in BAMs exposed to Lkt was assessed in the presence of 1.2 mM extracellular Ca²⁺. At this extracellular Ca²⁺ concentration, a Lkt concentration of >50 U/ml produced an elevation of [Ca²⁺]_i which was beyond the linear range of the camera. Therefore, in subsequent experiments, we determined the [Ca²⁺]_i response to Lkt concentrations of ≤50 U/ml. In order to exclude the effect of any postpurification LPS contamination in the Lkt preparations, purified Lkt fractions were incubated with 10 μg of polymyxin B per ml for 30 min on ice prior to use in all experiments involving Lkt. This concentration of polymyxin B was found to be sufficient to block the [Ca²⁺]_i response of BAMs to 100 ng of LPS per ml.

Concentration dependence of Lkt effects.

The cells were first washed with HBSS for 3 min to measure the basal [Ca²⁺]_i and then exposed to 1, 5, and 50 U of Lkt per ml. The duration of exposure to each of the Lkt concentrations was at least 2 min or after the [Ca²⁺]_i response reached a steady-state value. After exposure to 50 U of Lkt per ml, the cells were washed with HBSS for 2 min.

Effects of extracellular Ca²⁺ and nifedipine on the [Ca²⁺]_i response to Lkt.

The cells were first perfused with HBSS for 3 min, followed by nominally Ca²⁺-free HBSS for an additional 3 min and then to 50 U/ml Lkt in the same buffer. The cells were subsequently exposed to Ca²⁺-containing HBSS for 2 min, in the continued presence of Lkt, and the [Ca²⁺]_i response was measured. In other experiments, cells were preexposed to 3 μM nifedipine for 3 min and the [Ca²⁺]_i response to different concentrations of Lkt was measured.

Effects of neutralized and heat-inactivated Lkt on the [Ca²⁺]_i response.

Neutralized Lkt was prepared by incubating 50 U of bioactive Lkt with 0.5 μg of an anti-Lkt neutralizing monoclonal antibody (MAb 601; gift from S. Srikumaran, University of Nebraska, Lincoln) for 30 min on ice. In other experiments, bioactive Lkt was boiled for 30 min at 100°C. Loss of biological activity was confirmed in a XTT assay (26). The [Ca²⁺]_i responses to neutralized and heat-inactivated Lkt were measured.

Effect of LPS on the [Ca²⁺]_i response of BAMs.

Cells were washed with HBSS for 3 min and then exposed to 1 to 1,000 ng of P. haemolytica LPS per ml in the presence or absence of 5% FBS for an additional 5 min. To investigate the effect of extracellular Ca²⁺, the cells were washed with HBSS containing 1.2 mM Ca²⁺ for 3 min, followed by nominally Ca²⁺-free HBSS for an additional 3 min, and the [Ca²⁺]_i response to 100 ng of LPS per ml plus 5% FBS was measured. The [Ca²⁺]_i response to 1 ng of LPS per ml plus 5% FBS was measured in cells pretreated with 2 μM herbimycin A for ∼18 h.

Effect of pertussis toxin on Lkt- or LPS-induced elevation of [Ca²⁺]_i.

The [Ca²⁺]_i response to different concentrations of Lkt or 1 ng of LPS per ml plus 5% FBS was measured in cells pretreated with 200 ng of pertussis toxin per ml for ∼18 h.

Effects of phospholipase inhibitors and ETYA on Lkt-induced elevation of [Ca²⁺]_i.

The cells were first exposed to HBSS containing 2 μM methyl arachidonyl fluorophosphonate (MAFP) (PLA₂ inhibitor) or 2 μM U73122 (PLC inhibitor) or 10 μM 5,8,11,14-eicosatetraynoic acid (ETYA) (arachidonic acid analog) for 5 min, followed by different concentrations of Lkt for an additional 3-min period in the continued presence of the various agents, and the [Ca²⁺]_i response was measured.

Effects of U73122 and ETYA on LPS-induced elevation of [Ca²⁺]_i.

The cells were first exposed to HBSS containing 2 μM U73122 or 10 μM ETYA for 5 min, followed by 1 ng of LPS per ml plus 5% FBS for an additional 3-min period in the continued presence of these agents, and the [Ca²⁺]_i response was measured.

Materials.

DMEM, HBSS, antibiotics, and FBS were purchased from GIBCO/BRL (Grand Island, N.Y.). MAFP, herbimycin A, and pertussis toxin were purchased from Biomol (Plymouth Meeting, Pa.). U73122 was obtained from Research Biochemicals International (Natick, Mass.). ETYA was a gift from Hoffman-La Roche Laboratories (Nutley, N.J.). Nifedipine, HEPES, and other chemicals were obtained from Sigma Chemical Co. (St. Louis, Mo.). Fura-2/AM in dry DMSO was purchased from Molecular Probes (Eugene, Oreg.). Stock solutions of nifedipine and ETYA were made in absolute alcohol, and stock solutions of MAFP, herbimycin A, and U73122 were made in DMSO. The stock solutions were diluted in the working solution at least 1,000-fold. Fifty percent inhibitory concentrations of MAFP (2 μM [17]), ETYA (10 μM [2]), and U73122 (2 μM [31]) were used. The concentrations of these agents as well as of nifedipine were found to maximally inhibit Lkt-induced release of lactate dehydrogenase in a cytotoxicity assay (13).

Data analysis.

The F₃₄₀/F₃₈₀ ratios were used as an index of [Ca²⁺]_i, as described in an earlier publication (15). We found that the amplitude (difference between the prestimulus basal level and the peak of the response) of the elevation in [Ca²⁺]_i to Lkt or LPS varied for different cells and experiments. Therefore, the area under the [Ca²⁺]_i curve was used for comparison. In assessing the integrated [Ca²⁺]_i responses in these experiments, the period of integration was similar across all comparisons within a given protocol. The elevation of [Ca²⁺]_i by LPS, unlike by Lkt, returned to baseline levels even in the continued presence of this agent. In many cells, the basal [Ca²⁺]_i following the LPS response was below the preexposure level. To minimize the effect of this change in the basal [Ca²⁺]_i on the integrated [Ca²⁺]_i response to LPS, the period of integration was kept to the actual duration of the response. The integrated [Ca²⁺]_i response is a measure of the total Ca²⁺ released during the period of inquiry. The integrated [Ca²⁺]_i responses were compared by using unpaired Student’s t test. Outlier values were determined as those deviating from the mean value by more than 2 standard deviations. These values were excluded from the analyses (51 of 1,114 cells studied). Statistical significance was tested with P of <0.05. A P value of <0.05 was considered statistically significant.

From each coverslip, 15 to 20 cells were sampled. For each protocol, three to six different coverslips were used. The macrophages used in the present study were obtained from five healthy calves. BAMs from at least three animals were used in each of the experimental protocols described.

RESULTS

Concentration dependence of Lkt effects.

Exposure of BAMs to Lkt resulted in an elevation of [Ca²⁺]_i in a concentration-dependent fashion (Fig. 1; also see Fig. 7). Unlike the responses to 1 and 5 U/ml, the [Ca²⁺]_i response to 50 U/ml was rapid in onset, reaching a peak value within 60 s of exposure, sustained, and not reversible on washing with HBSS.

FIG. 1.

Trace showing the effects of different Lkt concentrations on [Ca²⁺]_i elevation in fura 2-loaded BAMs. Note the rapid [Ca²⁺]_i response to 50 U of Lkt per ml. Lkt effects were studied in the presence of 10 μg of polymyxin B per ml to block the effects of any contaminating LPS in the Lkt preparations used. The cells were exposed to 1, 5, and 50 U of Lkt per ml in a cumulative manner, i.e., no washing between exposures (n = 80 cells).

FIG. 7.

Integrated [Ca²⁺]_i responses of BAMs to 1, 5, and 50 U of Lkt per ml in the presence or absence of the various inhibitors. The responses to all three concentrations of Lkt are significantly inhibited by nifedipine (NFD), pertussis toxin (PTX), the PLA₂ inhibitor MAFP, the arachidonic acid analog ETYA, and the PLC inhibitor U73122.

Effects of extracellular Ca²⁺ and nifedipine on the [Ca²⁺]_i response to Lkt.

In order to assess whether Lkt-induced elevation of [Ca²⁺]_i required the presence of extracellular Ca²⁺, BAMs were incubated in nominally Ca²⁺-free HBSS before exposure to Lkt. Exposure of BAMs to Lkt (1 to 50 U/ml) in nominally Ca²⁺-free HBSS did not result in any elevation of [Ca²⁺]_i (Fig. 2A). However, return to Ca²⁺-containing medium in the presence of Lkt resulted in an elevation of [Ca²⁺]_i.

FIG. 2.

Representative traces showing the effect of extracellular Ca²⁺ removal (A) or nifedipine (B) on Lkt-induced [Ca²⁺]_i elevation in fura 2-loaded BAMs. In BAMs exposed to nominally Ca²⁺-free HBSS, there is no elevation of [Ca²⁺]_i to Lkt, even up to 50 U/ml. Subsequent exposure to Lkt in Ca²⁺-containing HBSS resulted in an elevation of [Ca²⁺]_i, as shown in panel A. In BAMs preexposed to nifedipine to block voltage-gated channels, the responses to 1 and 5 U of Lkt per ml are blocked, while the response to 50 U of Lkt per ml is significantly attenuated, as shown in panel B. In panels A and B, 64 and 66 cells, respectively, were used.

Nifedipine was used to block Ca²⁺ influx through voltage-gated L-type channels. In cells preexposed to nifedipine, the [Ca²⁺]_i response to 1 and 5 U of Lkt per ml was blocked, but the response to 50 U of Lkt per ml was significantly attenuated (Fig. 2B; also see Fig. 7).

Effects of neutralized and heat-inactivated Lkt on the [Ca²⁺]_i response.

To determine the specificity of Lkt effects, the [Ca²⁺]_i response of BAMs to neutralized or heat-inactivated Lkt was studied. Exposure to neutralized Lkt did not alter the basal [Ca²⁺]_i (Fig. 3A and B). Subsequent exposure to biologically active Lkt to ascertain viability of the cells resulted in an elevation of [Ca²⁺]_i. However, the integrated [Ca²⁺]_i response was significantly lower than the control Lkt response (compare Fig. 3B with Fig. 7). Heat-inactivated Lkt also did not elicit a [Ca²⁺]_i response, even at 50 U/ml (Fig. 3C and D). Subsequent exposure to biologically active Lkt caused a [Ca²⁺]_i elevation. The integrated [Ca²⁺]_i response to heat-inactivated Lkt was lower than the control Lkt response (compare Fig. 3D with Fig. 7).

FIG. 3.

(A) Representative trace shows that neutralized Lkt (50 U/ml) does not elicit a [Ca²⁺]_i response. After the cells were washed with HBSS, subsequent exposure to 50 U of bioactive Lkt per ml elicits an elevation of [Ca²⁺]_i. (B) Integrated [Ca²⁺]_i response to neutralized and bioactive Lkt in BAMs, represented as the area under the F₃₄₀/F₃₈₀ curve (n = 30 cells). (C) Representative trace shows that heat-inactivated Lkt (50 U/ml) does not elicit a [Ca²⁺]_i elevation, while subsequent exposure to 5 and 50 U of bioactive Lkt per ml, following a brief period of washing with HBSS, elicits an elevation of [Ca²⁺]_i. (D) Integrated [Ca²⁺]_i response to heat-inactivated and bioactive Lkt in BAMs, represented as the area under the F₃₄₀/F₃₈₀ curve. Note that the responses to bioactive Lkt are significantly attenuated than those of the controls (see Fig. 7) in these cells (n = 42 cells).

Effect of LPS on [Ca²⁺]_i elevation in BAMs.

LPS elicited a nonsustained elevation of [Ca²⁺]_i at all concentrations (1 to 1,000 ng/ml) only in the presence of FBS (Fig. 4A). In the absence of FBS, there was no elevation of [Ca²⁺]_i in response to LPS (up to 1,000 ng/ml) (Fig. 4B). The magnitude of the [Ca²⁺]_i elevation to 1 or 100 ng of LPS per ml was similar (Fig. 4C). In the presence of FBS, LPS induced an elevation of [Ca²⁺]_i, even in nominally Ca²⁺-free medium. However, this response was significantly lower than the control LPS response (Fig. 4C).

FIG. 4.

[Ca²⁺]_i response in BAMs to LPS. (A) Trace showing the response to 1 ng of LPS per ml in the presence of 5% FBS (n = 50 cells). (B) Representative trace showing no elevation of [Ca²⁺]_i even up to 1 μg of LPS per ml in the absence of FBS (n = 50 cells). Subsequent addition of 5% FBS resulted in an elevation of [Ca²⁺]_i. (C) Integrated [Ca²⁺]_i response of BAMs to LPS (1 or 100 ng/ml) (n = 129 cells) and the response to 100 ng of LPS per ml in nominally Ca²⁺-free HBSS (n = 62 cells). Note that there is no significant difference in the responses to the two concentrations of LPS. However, the response in nominally Ca²⁺-free HBSS is significantly attenuated. The asterisk denotes statistical significance (P < 0.05).

In order to determine the role of protein tyrosine kinase, BAMs were preexposed to herbimycin A for ∼18 h, and LPS-induced [Ca²⁺]_i elevation was measured. Herbimycin A significantly inhibited LPS-induced elevation of [Ca²⁺]_i (Fig. 5).

FIG. 5.

Integrated [Ca²⁺]_i response of BAMs to LPS in the presence or absence of various inhibitors. The integrated [Ca²⁺]_i response to 1 ng of LPS per ml plus 5% FBS is not affected by prior exposure to pertussis toxin (PTX) (n = 101 cells) or ETYA (n = 30 cells). However, preexposure to U73122 (n = 89 cells) or herbimycin A (Her A) (n = 93 cells) inhibits the responses to LPS significantly (P < 0.05). The period of integration is the actual duration of the response. An asterisk denotes statistical significance (P < 0.05).

Effects of pertussis toxin on Lkt- and LPS-induced elevation of [Ca²⁺]_i.

To assess whether Lkt or LPS effects are mediated through G_i- or G_o-type G proteins, BAMs were treated for ∼18 h with pertussis toxin before exposure to Lkt or LPS to study the [Ca²⁺]_i response. Pertussis toxin abolished the [Ca²⁺]_i response to 1 and 5 U of Lkt per ml, and the response to 50 U of Lkt per ml was significantly attenuated (Fig. 6A and 7). On the other hand, LPS-induced elevation of [Ca²⁺]_i was not affected by pertussis toxin (Fig. 5).

FIG. 6.

Representative traces showing the effects of pertussis toxin, MAFP, ETYA, and U73122 on Lkt-induced elevation of [Ca²⁺]_i in BAMs. (A) In BAMs treated with 200 ng of pertussis toxin per ml for 18 h, the [Ca²⁺]_i responses to 1 and 5 U of Lkt per ml are blocked, while the response to 50 U of Lkt per ml is attenuated (n = 71 cells). (B) In BAMs exposed to 2 μM MAFP, the [Ca²⁺]_i elevation to all concentrations of Lkt is blocked (n = 51 cells). The [Ca²⁺]_i response to 50 U of Lkt per ml is only partially restored on washing the cells with HBSS to remove the inhibitor. (C) Exposure to 2 μM U73122 blocks the elevation of [Ca²⁺]_i to all Lkt concentrations (n = 92 cells). The [Ca²⁺]_i response to 50 U of Lkt per ml is only partially restored on washing the cells with HBSS to remove the inhibitor. (D) Exposure to 10 μM ETYA blocks the [Ca²⁺]_i elevation in response to 1 and 5 U of Lkt per ml and attenuates the response to 50 U of Lkt per ml (n = 56 cells).

Effects of MAFP, ETYA, and U73122 on Lkt-induced elevation of [Ca²⁺]_i.

The role of arachidonic acid in mediating Lkt-induced elevation of [Ca²⁺]_i was assessed by inhibiting activity of phospholipases and by inhibiting arachidonic acid metabolism with the arachidonic acid analog ETYA. Preexposure to MAFP, the PLA₂ inhibitor, blocked the rise in [Ca²⁺]_i in response to 1 and 5 U of Lkt per ml and significantly attenuated the response to 50 U of Lkt per ml (Fig. 6B and 7). On washing the cells with HBSS following exposure to MAFP, the [Ca²⁺]_i response to 50 U of Lkt per ml was only partially restored (Fig. 6B). Preexposure to the PLC inhibitor U73122 blocked the elevation of [Ca²⁺]_i to all concentrations of Lkt (Fig. 6C and 7). ETYA blocked the [Ca²⁺]_i response to 1 and 5 U of Lkt per ml and significantly attenuated the response to 50 U of Lkt per ml (Fig. 6D and 7).

Effects of ETYA and U73122 on LPS-induced elevation of [Ca²⁺]_i.

Preexposure of BAMs to ETYA had no significant effects on LPS-induced elevation of [Ca²⁺]_i (Fig. 5), suggesting that arachidonic acid metabolites do not mediate the [Ca²⁺]_i response. However, the PLC inhibitor U73122 attenuated LPS-induced elevation of [Ca²⁺]_i (Fig. 5), further supporting a mechanism of [Ca²⁺]_i elevation by LPS which is different from that of Lkt.

DISCUSSION

In this study, we examined the effects of P. haemolytica serotype 1-derived Lkt and LPS on [Ca²⁺]_i levels and the mechanisms underlying the [Ca²⁺]_i regulation in BAMs. The results show that Lkt-induced elevation of [Ca²⁺]_i is concentration dependent, requires extracellular Ca²⁺, and is brought about by influx through voltage-gated L-type channels. Lkt effects on [Ca²⁺]_i elevation are mediated through a G-protein-coupled receptor, resulting in the activation of PLA₂ and PLC and production of arachidonic acid. On the other hand, LPS induces an elevation of [Ca²⁺]_i in the absence of extracellular Ca²⁺, suggesting Ca²⁺ release from intracellular stores. Strict serum requirement for LPS effects indicates the necessity for a LPS binding protein (LBP)-CD14-coupled signaling mechanism involving tyrosine phosphorylation and PLC activation.

Previous studies have indicated that activation of bovine neutrophils by P. haemolytica Lkt leads to elevation of [Ca²⁺]_i (22), an oxidative burst (19), and production of several lipid mediators (6, 11) and proinflammatory cytokines (43). Lkt-induced [Ca²⁺]_i elevation in bovine neutrophils and bovine lymphoma (BL-3) cells is dependent on extracellular Ca²⁺ and appears to be via voltage-gated channels (9, 22). RTX toxins derived from P. haemolytica and A. actinomycetemcomitans are known to form pores and increase cation conductance selectively in target cells. For example, exposure of susceptible target cells to Lkt of A. actinomycetemcomitans leads to rapid membrane depolarization and Ca²⁺ influx (33). Formation of cation-selective channels in lipid bilayer membranes by RTX toxins of A. pleuropneumoniae has also been reported (20). The findings of the present study not only confirm earlier reports of selective increase in Ca²⁺ permeability to Lkt but also provide evidence for the mechanisms by which Lkt and LPS elevate [Ca²⁺]_i in BAMs. The specificity of Lkt effects is demonstrated by the absence of a [Ca²⁺]_i response to neutralized or heat-inactivated Lkt. Presence of a [Ca²⁺]_i response to polymyxin B-treated Lkt indicates that the effects are indeed attributable to Lkt and not to LPS contamination. Lkt effects are inhibited by pertussis toxin, indicating the involvement of G_i- or G_o-protein-coupled receptors in elevation of [Ca²⁺]_i. This is the first demonstration of the involvement of G-protein-coupled signaling mechanism in Lkt-mediated [Ca²⁺]_i elevation in BAMs.

LPS-induced activation of alveolar macrophages involves several transmembrane signaling mechanisms (5, 32, 34, 41). LPS causes activation of phospholipases C, D, and A₂, resulting in the production of various metabolites (4, 10, 24). Some of the proposed models have invoked either a G-protein- or tyrosine kinase-coupled activation of phospholipases by LPS in macrophages (29, 30). However, this pathway of intracellular signaling may be mediated by the lipid A receptor, rather than the LBP-CD14 complex (21). Evidence for pertussis toxin-sensitive, G-protein-coupled signaling by LPS in macrophages from other species has been obtained (29). However, Jian et al. (14) have shown that superoxide production by LPS in BAMs requires serum, is mediated by tyrosine kinases and elevation of [Ca²⁺]_i, and does not rely on G-protein-mediated signaling. In macrophages from other species, LPS-CD14 interaction is also known to result in the activation of protein tyrosine kinases (35). The results of the present study provide evidence for tyrosine phosphorylation in LPS-mediated elevation of [Ca²⁺]_i through a non-G_i- or G_o-coupled activation of PLC. The strict requirement for serum for the [Ca²⁺]_i response to LPS and the ability of very low concentrations of LPS (1 to 10 ng/ml) to elicit a response in BAMs clearly point to a LBP-CD14-dependent signaling mechanism. Although [Ca²⁺]_i elevation induced by LPS is not affected by pertussis toxin, the role of other G proteins cannot be ruled out.

Our results show that LPS-induced elevation of [Ca²⁺]_i is not blocked by removal of extracellular Ca²⁺, indicating mobilization from intracellular stores. Similar findings have been reported in murine (23, 24) and rat peritoneal macrophages (16) in response to LPS. LPS-mediated intracellular Ca²⁺ release in BAMs does not involve arachidonic acid, since the arachidonic acid analog ETYA had no significant effects on the elevation of [Ca²⁺]_i. In macrophages from other species, IP₃ released from PLC-induced hydrolysis of phosphatidylinositol bisphosphate (PIP₂) mediates intracellular Ca²⁺ mobilization by LPS (16, 23, 24). Therefore, the effects of U73122 on LPS- and Lkt-induced [Ca²⁺]_i release in BAMs should be interpreted in the context of the role of arachidonic acid in these responses. Inhibition of LPS-mediated [Ca²⁺]_i release by the PLC inhibitor is consistent with inhibition of PIP₂ hydrolysis and decreased IP₃ production, since arachidonic acid does not appear to have any role in the release of intracellular Ca²⁺.

DAG, a product of PLC, can be converted to arachidonic acid through the DAG lipase pathway (1). DAG is also known to activate protein kinase C (PKC) in a variety of cell systems (3). Therefore, the inhibitory effects of U73122 on Lkt-induced [Ca²⁺]_i elevation most likely reflects inhibition of arachidonic acid formation and/or decreased PKC activation of phospholipases (8). A detailed elucidation of these alternative pathways during Lkt stimulation of BAMs is clearly beyond the scope of the present study.

Previous studies from our laboratory have suggested that Ca²⁺ is required for proinflammatory cytokine gene expression in Lkt-stimulated BAMs, since chelation of extracellular Ca²⁺ by EGTA inhibited tumor necrosis factor alpha and interleukin 1β mRNA expression (43). Another study has shown that buffering the [Ca²⁺]_i with a chelator decreased LPS-induced tissue factor expression in vascular endothelial cells (40). These data provide clear evidence for the importance of [Ca²⁺]_i in the expression of proinflammatory cytokine gene and tissue factor. However, it is not known whether LPS-induced [Ca²⁺]_i elevation is necessary for cytokine gene expression in BAMs.

Our results indicate that Lkt and LPS induce elevation of [Ca²⁺]_i in BAMs through different signaling mechanisms (see Fig. 8 for a proposed model). Lkt-induced [Ca²⁺]_i elevation involves a G-protein-coupled activation of L-type Ca²⁺ channels. G proteins, being coupled to PLA₂ and PLC, regulate arachidonic acid release and the gating of Ca²⁺ channels. However, the arachidonic acid metabolites involved in the regulation of Ca²⁺ influx in BAMs are not known. DAG, a product of PLC activity, can directly activate PKC, which in turn may regulate phospholipase activity (8). Although there is no direct evidence for intracellular Ca²⁺ release during Lkt stimulation, the dependence of [Ca²⁺]_i elevation on extracellular Ca²⁺ appears to rule out any role for such a mechanism. In contrast, LPS-mediated [Ca²⁺]_i elevation through a CD14-dependent pathway involves intracellular mobilization, does not require activation of G_i or G_o, but involves tyrosine phosphorylation. Whether tyrosine kinase regulates PLC activation and/or IP₃-induced Ca²⁺ release from the endoplasmic reticulum during LPS stimulation of BAMs is unclear. The differences in the signaling mechanisms associated with Lkt and LPS may underlie the reported differences in the kinetics of proinflammatory cytokine gene expression in BAMs (42, 43).

FIG. 8.

Proposed model for Ca²⁺ signaling in response to P. haemolytica-derived Lkt and LPS in BAMs. The LPS-LBP complex interacts with CD14, resulting in the release of intracellular Ca²⁺ from the endoplasmic reticulum. This pathway does not involve activation of pertussis toxin-sensitive G proteins but requires activation of protein tyrosine kinase (PTK), since it is inhibited by herbimycin A. Modulation of LPS-induced [Ca²⁺]_i elevation in BAMs by PLC inhibition suggests the involvement of IP₃-induced Ca²⁺ release from the endoplasmic reticulum. Lkt elevates [Ca²⁺]_i through G-protein-coupled activation of Ca²⁺ influx via L-type channels. This G protein is coupled to PLA₂ and PLC. Arachidonic acid (AA) derived from PLA₂ activation gates the Ca²⁺ channels. PLC activation by Lkt may result in AA formation through the conversion of DAG by DAG lipase and/or by regulation of phospholipase activity by PKC, which is activated by DAG.