Mannheimia haemolytica leukotoxin activates a nonreceptor tyrosine kinase signaling cascade in bovine leukocytes, which induces biological effects

Abstract

The leukotoxin (LktA) produced by Mannheimia haemolytica binds to bovine lymphocyte function-associated antigen 1 (LFA-1) and induces biological effects in bovine leukocytes in a cellular and species-specific fashion. We have previously shown that LktA also binds to porcine LFA-1 without eliciting any effects. These findings suggest that the specificity of LktA effects must entail both binding to LFA-1 and activation of signaling pathways which are present in bovine leukocytes. However, the signaling pathways leading to biological effects upon LktA binding to LFA-1 have not been characterized. In this context, several reports have indicated that ligand binding to LFA-1 results in activation of a nonreceptor tyrosine kinase (NRTK) signaling cascade. We designed experiments with the following objectives: (i) to determine whether LktA binding to LFA-1 leads to activation of NRTKs, (ii) to examine whether LktA-induced NRTK activation is target cell specific, and (iii) to determine whether LktA-induced NRTK activation is required for biological effects. We used a biologically inactive mutant leukotoxin (DeltaLktA) for comparison with LktA. Our results indicate that LktA induces tyrosine phosphorylation (TP) of the CD18 tail of LFA-1 in bovine leukocytes. The DeltaLktA mutant does not induce TP of the CD18 tail, albeit binding to bovine LFA-1. LktA-induced TP of the CD18 tail was attenuated by an NRTK inhibitor, herbimycin A; a phosphatidylinositol 3'-kinase (PI 3-kinase) inhibitor, wortmannin; and a Src kinase inhibitor, PP2, in a concentration-dependent manner. Furthermore, LktA induces TP of the CD18 tail in bovine, but not porcine, leukocytes. Moreover, LktA-induced intracellular calcium ([Ca2+]i) elevation was also inhibited by herbimycin A, wortmannin, and PP2. Thus, our data represent the first evidence that binding of LktA to bovine LFA-1 induces a species-specific NRTK signaling cascade involving PI 3-kinase and Src kinases and that this signaling cascade is required for LktA-induced biological effects.

FIG. 1

Flow cytometric detection of β2 integrins in BAMs. Cells were incubated with various anti-β2 integrin MAbs or a control MAb and then incubated with FITC-labeled anti-mouse secondary antibody as described in Materials and Methods; results are expressed as MFI. BAMs expressed high levels of CD11a and CD18 and low levels of CD11b and CD11c. Data are from one representative experiment of three experiments performed.

FIG. 2

LktA binding to LFA-1 is not influenced by herbimycin A, wortmannin, PP2, or PP3 as determined by flow cytometry. Cells were preincubated with medium alone or medium containing herbimycin A (Her A; 1 μM), wortmannin (Wort; 5 μM), PP2 (5 μM), or PP3 (5 μM), washed, and exposed to LktA. Thereafter, the cells were washed and incubated with anti-LFA-1 (CD11a/CD18) MAb followed by incubation with FITC-labeled goat anti-mouse secondary antibody. Results are expressed as the percent LktA binding to LFA-1, according to the following formula: % LktA binding to LFA-1 = [(MFI of anti-LFA-1 MAb − MFI of anti-LFA-1 MAb after pretreatment with LktA)/MFI of anti-LFA-1 MAb] × 100. Means and standard errors (indicated by error bars) of three experiments are shown.

FIG. 3

Biologically inactive ΔLktA binds to bovine LFA-1. BAM lysates were incubated with ΔLktA- or BSA-coated beads for 15 h at 4°C as described in Materials and Methods. Bound proteins from the beads were then eluted, electrophoresed on an SDS–4-to-15%-gradient polyacrylamide gel, transferred onto a PVDF membrane, and analyzed by Western blotting using anti-CD18 (BAQ30A) (A) or anti-CD11a (MUC76A) (B) MAb. Cell lysates from BAMs show 90- and 85-kDa CD18 bands and a 180-kDa CD11a band (panels A and B, lane 1). The eluant from ΔLkt-coated beads that were reacted with BAM lysates contained 90- and 85-kDa CD18 bands and a 180-kDa CD11a band (panels A and B, lane 2). In the eluant from Lkt-coated beads preincubated with anti-Lkt MAb (MAb601) before adding BAM lysates or in the eluant from BSA-coated beads incubated with BAM lysates, no CD18 bands or CD11a bands were observed (panels A and B, lanes 3 and 4). The data are from one representative experiment of three experiments performed.

FIG. 4

LktA interaction with BAMs results in TP of the CD18 tail. BAMs were incubated for 2 min at 37°C with LktA (lane 1), no LktA (lane 2), ΔLktA (lane 3), LktA preincubated with anti-LktA (MAb 601; lane 4), or LPS (1 μg/ml; lane 5). Cell lysates were immunoprecipitated with anti-CD18 MAb (BAT75A; lanes 1 to 5) or an irrelevant control antibody (MOPC21; lane 6), electrophoresed on an SDS–4-to-15%-gradient polyacrylamide gel, and transferred onto a PVDF membrane. The blot was developed with antiphosphotyrosine MAb (panels A and B). The membrane was stripped and reprobed sequentially with anti-CD18 (BAQ30A) (C) or anti-CD11a (MUC76A) (D) MAb. Only the biologically active LktA induced TP of the CD18 tail, but not of the CD11a tail, as indicated by an arrow on the left of panel A, lane 1. The data are from one representative experiment of three experiments performed. Molecular masses are shown in kilodaltons.

FIG. 5

LktA-induced TP of the CD18 tail is blocked by anti-LFA-1 (CD11a/CD18) MAbs. BAMs were preincubated with 5 μg of various anti-β2 integrin MAbs for 30 min at 37°C before exposure to LktA for 2 min at 37°C. Cell lysates were immunoprecipitated with anti-CD18 MAb (BAT75A), electrophoresed on an SDS–4-to-15%-gradient polyacrylamide gel and transferred onto a PVDF membrane. The blot was first developed with antiphosphotyrosine MAb (A), stripped, and reprobed with anti-CD18 (BAQ30A) (B). Anti-CD18 and anti-CD11a (lanes 5 and 6), but not anti-CD11b or anti-CD11c (lanes 3 and 4), blocked LktA-induced TP of the CD18 tail. The arrow at the left of panel A indicates the position of tyrosine-phosphorylated CD18. The data shown are from one representative experiment of three experiments performed.

FIG. 6

LktA-induced TP of the CD18 tail is inhibited by herbimycin A. BAMs were preincubated with 0.06 to 1 μM herbimycin A for 10 min at 37°C before exposure to LktA for 2 min at 37°C. Cell lysates were immunoprecipitated with anti-CD18 MAb (BAT75A), electrophoresed on an SDS–4-to-15%-gradient polyacrylamide gel, and transferred onto a PVDF membrane. The blot was first developed with antiphosphotyrosine MAb (A), stripped, and reprobed with anti-CD18 MAb (BAQ30A) (B). The arrow at the left of panel A indicates the position of the tyrosine-phosphorylated CD18. The data shown are one representative experiment of four experiments performed.

FIG. 7

LktA-induced TP of the CD18 tail is inhibited by wortmannin and PP2, but not by PP3. BAMs were preincubated with 0.65 to 10 μM wortmannin, PP2, or PP3 before exposure to LktA for 2 min at 37°C. Cell lysates were immunoprecipitated with the anti-CD18 MAb (BAT75A), electrophoresed on an SDS–4-to-15%-gradient polyacrylamide gel, and transferred onto a PVDF membrane. The blot was first developed with antiphosphotyrosine MAb (A), stripped, and reprobed with anti-CD18 MAb (BAQ30A) (B). Wortmannin and PP2, but not PP3, inhibited TP in a concentration-dependent manner. The arrow at the left of panel A indicates the position of tyrosine-phosphorylated CD18. The data shown are from one representative experiment of three experiments performed.

FIG. 8

LktA interaction with PAMs results in no TP of the CD18 tail. However, anti-porcine CD11a (MUC76A; lane 4)—but not anti-bovine CD18 (BAT75A, lane 3), anti-canine CD11a (R3.1, lane 5), or anti-canine CD18 (R15.7, lane 6)—induces TP of the CD18 tail in PAMs. Cells were incubated with 50 U of LktA per ml or 5 μg of various MAbs for 2 min at 37°C. Cell lysates were immunoprecipitated with the anti-CD18 MAb (BAT75A), electrophoresed on an SDS–4-to-15%-gradient polyacrylamide gel, and transferred onto a PVDF membrane. The blot was first developed with antiphosphotyrosine MAb (A), stripped, and reprobed with anti-CD18 MAb (BAQ30A) (B). The arrow at the left of panel A indicates the position of tyrosine-phosphorylated CD18. The data shown are from one representative experiment of five experiments performed.

FIG. 9

Integrated LktA-induced [Ca²⁺]_i elevation in BAMs in the presence or absence of herbimycin A, wortmannin, PP2, or PP3. Cells were preincubated with different concentrations of herbimycin A, wortmannin, PP2, and PP3 for 10 min at 37°C before exposure to 50 U of LktA per ml. Cells were loaded with Fura-2-AM and the [Ca²⁺]_i elevation was measured at nanomolar levels. Means and standard errors (indicated by error bars) of four experiments are shown. At least 120 cells were included in each experiment. Values that are significantly different from the control value (P < 0.05) are indicated by asterisks.