IAI FigSearch
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
IAI.00808-07v1
75/10/5004    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dassanayake, R. P.
Right arrow Articles by Srikumaran, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dassanayake, R. P.
Right arrow Articles by Srikumaran, S.
Infection and Immunity, October 2007, p. 5004-5010, Vol. 75, No. 10
0019-9567/07/$08.00+0     doi:10.1128/IAI.00808-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Monomeric Expression of Bovine ß2-Integrin Subunits Reveals Their Role in Mannheimia haemolytica Leukotoxin-Induced Biological Effects{triangledown}

Rohana P. Dassanayake,1 Samuel K. Maheswaran,2 and Subramaniam Srikumaran1*

Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-7040,1 Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, Minnesota 551082

Received 12 June 2007/ Returned for modification 16 July 2007/ Accepted 1 August 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The ruminant-specific leukotoxin (Lkt) of Mannheimia haemolytica is the key virulence factor contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies by us and others indicate that M. haemolytica Lkt binds to CD18, the ß subunit of bovine ß2-integrins on leukocytes, and that the species specificity of Lkt-induced effects is resident in the ß subunit CD18 and not in the {alpha} subunit CD11. However, Lkt also binds to the CD11a subunit of LFA-1. Furthermore, antibodies specific for CD18 or CD11a inhibit signaling events leading to elevation of intracellular [Ca2+], tyrosine phosphorylation of the cytosolic domain of CD18, and cytolysis of bovine leukocytes. These observations underscore the need for further investigation to identify the precise subunit of bovine LFA-1 utilized by M. haemolytica Lkt as the functional receptor. For this purpose, monomeric bovine CD18 and CD11a and heterodimeric LFA-1 were expressed in the HEK-293 cell line by transfection, and the resulting transfectants were tested for susceptibility to Lkt-induced effects. All three transfectants effectively bound Lkt. However, Lkt-induced cytolysis was observed only with transfectants expressing monomeric bovine CD18 or LFA-1. Furthermore, intracellular [Ca2+] elevation following exposure to Lkt, which is a marker for postbinding signaling leading to cellular activation, was seen only with transfectants expressing monomeric bovine CD18 or LFA-1. These results clearly indicate that the bovine CD18 subunit of ß2-integrins is the functional receptor for M. haemolytica Lkt.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Mannheimia (Pasteurella) haemolytica serotype 1 is the primary bacterial pathogen of bovine pneumonic pasteurellosis, which is commonly known as shipping fever, causing extensive economic losses to the beef and dairy cattle industry in the United States and elsewhere (3, 4, 6, 13, 19). The disease is an acute respiratory infection which results in severe fibrinous pleuropneumonia, often followed by the deaths of infected animals (19, 41). M. haemolytica is a gram-negative, nonmotile coccobacillus which is a commensal bacterium of tonsillar crypts and the upper respiratory tract of healthy cattle as well as many other ruminants. Various factors such as stress and concurrent viral or bacterial infections facilitate its colonization in the lower respiratory tract, resulting in the development of pneumonia (5). The bacterium produces several virulence determinants, of which leukotoxin (Lkt) and lipopolysaccharide are the major determinants that largely contribute to the pathogenesis of pneumonia (14, 15, 37). M. haemolytica Lkt is a calcium-dependent cytotoxin. It is a protein with an approximate molecular mass of 102 kDa, produced in high concentrations during the logarithmic growth phase (2, 19). It is a member of the repeats-in-toxin (RTX) family of pore-forming cytolysins that shows cell type as well as species specificity. The Lkt has narrow target cell specificity in that it interacts only with leukocytes from cattle, goats, sheep, and other ruminants (2, 4, 36). This observation has led to the hypothesis that species and cell-specific effects are mediated through a specific receptor which is unique to ruminant leukocytes.

We and others have independently shown that the cytotoxic effect of Lkt on bovine and ovine leukocytes is mediated by Lkt-ß2-integrin interactions (1, 7-10, 17, 23, 24, 26, 40). ß2-Integrins are leukocyte-specific integrins that are critical for homing leukocytes to the sites of inflammation, phagocytosis, antigen presentation, and cytotoxicity (11, 27). They are expressed on the cell surface as heterodimeric glycoproteins composed of the {alpha} subunit CD11 and the ß subunit CD18. These subunit associations result in the expression of four different ß2-integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (CR4), and CD11d/CD18 (11). CD11d/CD18 has not been characterized well in the ruminants (31). Since Lkt binds to CD18 and species-specific susceptibility to Lkt-induced effects is resident in the CD18 subunit, a consensus within the field is that the CD18 subunit of ß2-integrin(s) is the receptor for Lkt. Indeed, in our previous studies, the recombinant expression of bovine or ovine CD18 in an Lkt nonsusceptible cell line rendered it susceptible to Lkt-induced cytolysis, indicating that CD18 is necessary and sufficient to mediate Lkt-induced cytolysis of target cells (7, 8, 26). However, in these studies, CD18 was expressed as a heterodimer with murine CD11a, which precluded the elucidation of the role of CD11a, if any, in Lkt-induced cytolysis of ruminant leukocytes. In related studies, Lkt also bound to the CD11a subunit of LFA-1 (17). In addition, antibodies specific for CD18 or CD11a were equally effective in inhibiting signaling events, leading to the elevation of intracellular [Ca2+], tyrosine phosphorylation of the cytosolic domain of CD18, and cytolysis of bovine leukocytes (10, 18, 38). These discordant findings underscore the need for additional studies to identify the precise functional receptor to which Lkt binds and initiates signaling events leading to activation and cytolysis of bovine leukocytes.

Past studies have shown that the {alpha} subunit CD11 and the ß subunit CD18 have to associate with each other in order to be transported to and expressed on the leukocyte surface (20, 28). Recently, monomeric human integrins CD11a, CD11b, and CD18 have been successfully expressed in several nonhematopoietic cell lines such as human embryonic kidney (HEK)-293 and COS cells (22, 35, 42). Therefore, we took advantage of the ability of HEK-293 cells to express monomeric CD11a and CD18, as well as their nonsusceptibility to M. haemolytica Lkt-induced cytolysis to generate transfectants expressing monomeric bovine CD11a or CD18. We also generated an HEK-293 transfectant expressing heterodimeric CD11a/CD18 and used all three transfectants to elucidate the role of each subunit in Lkt-LFA-1 interactions.

The specific objectives of this study were to determine (i) if binding of Lkt to CD18 alone has any functional consequence, (ii) if CD18 dimerization with CD11a is required for the responsiveness to Lkt-induced biological effects, (iii) if binding of Lkt to CD11a has any functional consequence, and (iv) which one of these subunits serves as the functional receptor for M. haemolytica Lkt. Here we present data which demonstrate unequivocally that bovine CD18 is the functional receptor for M. haemolytica Lkt.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Cell lines and growth conditions. The HEK-293 cell line (ATCC CRL-1573) was maintained in complete Dulbecco's modified Eagle's growth medium (Invitrogen, Carlsbad, CA) containing 4 mM L-glutamine and 100 µg/ml normocin (Amaxa Inc., Gaithersburg, MD) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Atlanta Biologicals, Norcross, GA) at 37°C in a humidified atmosphere of 5% CO2. Transfectants stably expressing monomeric bovine CD11a or CD18 or heterodimeric LFA-1 were selected and maintained in complete growth medium supplemented with blasticidin (40 µg/ml for CD11a), G418 (Geneticin; 1.5 mg/ml for CD18), or both antibiotics (for LFA-1) (InvivoGen, San Diego, CA).

Monoclonal antibodies. Anti-human CD11a monoclonal antibody (MAb) HUH73A (immunoglobulin G1 [IgG1]), which cross-reacts with bovine CD11a, and anti-bovine CD18 MAb BAQ30A (IgG1) were obtained from Washington State University Monoclonal Antibody Center. The Lkt-neutralizing MAb MM601 (IgG1) and the Lkt-nonneutralizing MAb MM605 conjugated to fluorescein isothiocyanate (FITC) were used for Lkt neutralization and Lkt-CD11a/CD18 binding studies, respectively (12). The MAb 8G12 (IgG1) specific for bovine respiratory syncytial virus was obtained from the Department of Veterinary and Biomedical Sciences at the University of Nebraska-Lincoln and used as an isotype-matched control MAb (21).

Production of M. haemolytica Lkt. M. haemolytica Lkt was prepared from culture supernatants by using a previously described procedure (12). All experiments were performed with the same batch of Lkt.

Bovine CD11a and CD18 cDNAs. Bovine CD11a cDNA was made from total cellular RNA isolated from a bovine lymphoma cell line (BL-3; ATCC CRL-8037) using the Superscript III reverse transcription (RT)-PCR system according to the manufacturer's protocol (Invitrogen). Gene-specific primers were designed based on the available bovine CD11a sequence, and CD11a was amplified by PCR using high-fidelity PfuUltra II Fusion HS DNA polymerase as described by the manufacturer (Stratagene, La Jolla, CA). The PCR products obtained were cloned into the mammalian expression vector pcDNA6.2/GW/D-TOPO (Invitrogen) to yield pRD/Bo CD11a. The bovine cDNA for CD18 (34) was generously provided by Marcus Kehrli (NADC, Ames, IA), and subcloned into the mammalian expression vector pCI-neo to yield pMD1 (8).

Transfection of bovine CD11a and CD18 into HEK-293 cells. The mammalian expression vectors carrying cDNAs for bovine CD11a (pRD/Bo CD11a) and CD18 (pMD1) were transfected either individually or together into HEK-293 cells, using TransFast transfection reagent according to the manufacturer's protocol (Promega, Madison, WI). Two days posttransfection, cells were transferred into 75-cm2 tissue culture flasks containing the selection medium blasticidin (40 µg/ml for bovine [Bo] CD11a transfectants), G418 (1.5 mg/ml for Bo CD18 transfectants), and both antibiotics for Bo LFA-1 transfectants. Transfectants stably expressing monomeric bovine CD11a or CD18 or heterodimeric LFA-1 on their cell surface were evaluated by flow cytometric analysis with MAbs specific for bovine CD11a or CD18.

Flow cytometric analysis of cell surface expression of bovine CD11a and CD18. The transfectants were examined for surface expression of bovine CD11a or CD18 following labeling with appropriate MAbs, as previously described (7). Briefly, 2.5 x 105 bovine transfectants in 50 µl of fluorescence-activated cell sorter (FACS) buffer (3% horse serum and 0.01% sodium azide in phosphate-buffered saline [PBS]) were incubated with 50 µl of either the MAb BAQ30A (30 µg/ml) or HUH73A (30 µg/ml) on ice for 15 min. Following three washes in FACS buffer, the cells were incubated with 100 µl of FITC-conjugated goat anti-mouse Ig on ice for another 15 min (1:200 dilution; Caltech Laboratories, Burlingame, CA). The parent HEK-293 cell line was used as a negative control and was treated similarly with both antibodies. The cells were washed three times with FACS buffer, resuspended, and analyzed in a flow cytometer (FACSort; Becton-Dickinson Immunocytometry Systems, San Jose, CA). Transfectants expressing monomeric bovine CD11a or CD18 or heterodimeric LFA-1 were enriched by mini magnetic cell sorting columns following labeling of transfectants with anti-CD11a or anti-CD18 MAbs (30 µg/ml), followed by goat anti-mouse IgG conjugated to superparamagnetic microbeads (20 µl) according to the manufacturer's protocol (Miltenyi Biotech Inc., Auburn, CA).

Lkt-binding assay. The ability of M. haemolytica Lkt to bind transfectants was evaluated by flow cytometry as previously described (7). Briefly, 5 x 105 bovine transfectants in 50 µl of colorless RPMI medium were incubated with 50 µl of Lkt (640 units per ml) on ice for 40 min, followed by fixation with 2% paraformaldehyde in PBS at 4°C for 10 min. Following three washes in FACS buffer, transfectants were incubated with 100 µl of Lkt-nonneutralizing MAb MM605-FITC (1:75 dilution) on ice for 15 min and then washed three times with FACS buffer. The HEK-293 parent cells incubated with Lkt, followed by MM605-FITC, served as the negative control for the Lkt-binding assay.

Determination of Lkt-induced cytolysis of bovine transfectants. The susceptibility of transfectants to M. haemolytica Lkt-induced cytolysis was determined by a previously described MTT (3-[4,5-dimethylthiazoyl-2-Yl]-2,5-diphenyl tetrazolium bromide [Sigma Chemical Co., St. Louis, MO]) dye reduction cytotoxicity assay (7, 8, 12). This assay measures the ability of the endoplasmic reticulum resident enzymes in viable cells to convert a tetrazolium dye into a purple formazan precipitate, which is later dissolved in cold acid isopropanol. The optical density (OD) of the end product, representing the intensity of the purple color developed, is directly proportional to the viability of the cells. The percentage of cytolysis was calculated as [1 – (OD of toxin-treated cells/OD of toxin-untreated cells)] x 100.

Inhibition of Lkt-induced cytolysis. The inhibition of Lkt-induced cytolysis of the transfectants by MAbs specific for CD18 or CD11a was determined by the cytotoxicity assay described above, with the exception that the cells were preincubated with the anti-CD18 MAb BAQ30A (50 µg/ml), the anti-CD11a MAb HUH73A (50 µg/ml), or the isotype-matched control MAb 8G12 (50 µg/ml) at 4°C for 40 min before incubation with Lkt. In order to demonstrate that the cytolytic effect on the transfectants was specifically induced by Lkt, the cytotoxicity assay was performed after the preincubation of Lkt with Lkt-neutralizing MAb MM601 (10 µg/ml) at 4°C for 40 min. The isotype-matched control MAb 8G12 served as the negative control. The percentage of inhibition of cytolysis was calculated [(1 – the percentage of cytolysis in the presence of antibody)/the percentage of cytolysis in the absence of antibody] x 100.

Determination of Lkt-induced intracellular [Ca2+] elevation. Intracellular [Ca2+] elevation in transfectants exposed to Lkt was measured by using the fluorescent calcium indicator Fluo-4-AM (Molecular Probes, Eugene, OR). Briefly, 2.5 x 105 transfectants or parent cells (HEK-293) were resuspended in 50 µl of Hank's balanced salt solution containing 1.25 mM CaCl2, 0.6 mM MgCl2, and 50 µl of 2x cell-permeant fluo-4-acetoxymethyl ester (Fluo-4-AM) and were incubated at 37°C for 30 min, followed by incubation at room temperature for another 30 min. Cells were incubated with Lkt (5 to 40 units) for 60 s, and intracellular [Ca2+] elevation was measured by flow cytometry. At least 10,000 cells were counted to evaluate the intracellular [Ca2+] elevation. Culture supernatant from the M. haemolytica Lkt deletion mutant (Lkt mutant strain) was used as the negative control (29).

Statistical analysis. Cytolysis of bovine transfectants and inhibition of cytolysis following preincubation of transfectants with MAbs were expressed as mean percentage of cytolysis or percentage of inhibition of cytolysis, with their corresponding standard deviations, respectively. Data were statistically analyzed by the Student t test, and P values were determined using Statistical Analysis System (version 8; SAS Institute Inc., Cary, NC) software. The term "significant" indicates a P value of less than 0.05.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Previous studies by us and others confirmed ß2-integrins as the receptors for Lkt on the leukocytes of cattle, bighorn sheep, and domestic sheep (1, 10, 17, 23, 24, 38, 40). In further studies in our laboratory, the recombinant expression of CD18 from cattle or domestic sheep or bighorn sheep rendered a Lkt-nonsusceptible murine cell line susceptible to Lkt-induced cytolysis (7, 8, 26), prompting us to propose that CD18 is necessary and sufficient to mediate Lkt-induced cytolysis of target cells. However, in these studies we could not definitively rule out a role for the {alpha} subunit CD11a in Lkt-induced cytolysis because the transfected CD18 was expressed as a heterodimer with murine CD11a. Therefore, in this study we developed transfectants which exhibit monomeric expression of either bovine CD11a or CD18 in order to clarify the role played by the individual subunits without the confounding effect of the other.

Transfectants express monomeric bovine CD11a or CD18 or LFA-1 on their surfaces. We selected the nonhematopoietic cell line HEK-293 for this study because of its nonsusceptibility to Lkt-induced cytolysis and its ability to express monomeric ß2-integrins on the cell surface (23, 35, 42). Transfectants coexpressing bovine CD11a and CD18 (Bo LFA-1) were obtained in much larger numbers (sixfold greater) than the transfectants expressing either subunit (Bo CD11a or Bo CD18). The coexpression of CD11a and CD18 on the cell surface of Bo LFA-1 transfectants was almost 1 log greater than the expression of CD11a and CD18 on Bo CD11a and Bo CD18 transfectants, respectively (data not shown). Furthermore, expression of the CD11a/CD18 heterodimer on Bo LFA-1 was stable for much longer periods than the expression of CD11a and CD18 on Bo CD11a and Bo CD18, respectively, necessitating frequent selective enrichment with appropriate MAb and magnetic sorting. Although either subunit can be expressed by itself on the cell surface of HEK-293 cells, these observations indicate that the association of CD11a and CD18 in the cytosol greatly enhances the transport and stable expression of both subunits on the cell surface. Since it is well established that CD11a and CD18 in leukocytes have to associate with each other to form a heterodimer in order to be transported to the surface of leukocytes (28), it is very likely that Bo LFA-1 is expressed as a heterodimer. This conclusion is supported by the coprecipitation of bovine CD18 with murine CD11a in an earlier study in our laboratory (8) and by the coprecipitation of human CD18 with CD11a or CD11b from human Mac1 transfectants by Solovjov et al. (35). It has been clearly demonstrated by a variety of methods that the individual human ß2-integrin subunits were not expressed on HEK-293 cells (35, 42) in association with subunits belonging to any other family of integrins endogenously expressed by these cells. Therefore, it is very likely that the monomeric bovine CD11a and CD18 subunits are transported to and expressed on the cell surface of HEK-293 cells by themselves, without association with any other endogenous integrin subunits. Furthermore, it is likely that the CD11a and CD18 subunits are expressed as monomers and not as homodimers, since the GFFKR motif on the CD18 cytosolic domain enables only the association of CD11a with CD18 and not the formation of homodimers (11, 20, 28). Transfectants coexpressing bovine CD11a and CD18 as a heterodimer (LFA-1) enabled us to compare Lkt binding, Lkt-induced cytolysis, and intracellular [Ca2+] elevation among the transfectants expressing the ß2-integrin subunits CD11a and CD18, either as monomers or as heterodimers.

Lkt binds to the transfectants expressing bovine CD11a and CD18. In order to determine the ß2-integrin subunit specificity of Lkt binding, the transfectants expressing monomeric bovine CD11a (Bo CD11a) or CD18 (Bo CD18) were subjected to flow cytometric analysis. Transfectants expressing both bovine CD11a and CD18 as a heterodimer (Bo LFA-1) were used as the positive control, and the parent cells (HEK-293) were used as the negative control for Lkt binding. As expected, Lkt bound to Bo LFA-1 (98%) but not to HEK-293 cells. Lkt also bound to both the Bo CD11a (82%) and Bo CD18 (92%) transfectants, indicating that Lkt does bind to bovine CD18 as well as to CD11a (Fig. 1). This was not an unexpected observation since our earlier studies have shown that Lkt-LFA-1 binding is inhibited by anti-CD11a or anti-CD18 MAbs (17). We have also demonstrated Lkt-CD11a and Lkt-CD18 binding by affinity chromatography-based binding assays with bovine alveolar macrophages and LFA-1 transductants (10, 38). Therefore, it is very likely that at least one or perhaps more Lkt-binding sites are present in the bovine CD11a and CD18 subunits.


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 1. M. haemolytica Lkt binds to transfectants expressing monomeric bovine CD11a or CD18 or heterodimeric LFA-1. The bovine transfectants Bo CD11a (panel A), Bo CD18 (panel B), and Bo LFA-1 (panel C) and HEK-293 parent cells (panels A to C) were incubated with Lkt, fixed with 2% paraformaldehyde, washed, and incubated with FITC-conjugated Lkt nonneutralizing MAb MM605. Results of one representative experiment out of three are shown.

 
Lkt induces cytolysis of transfectants expressing monomeric bovine CD18 but not CD11a. Lkt does bind to nonruminant leukocytes but does not induce cytolysis or intracellular [Ca2+] elevation (17), suggesting that Lkt binding itself is not sufficient to induce cytolysis. Therefore, it was of interest to determine whether Lkt binding to monomeric bovine CD11a expressed on Bo CD11a transfectants and monomeric bovine CD18 expressed on Bo CD18 transfectants lead to cytolysis of these cells. We subjected the Bo CD11a and Bo CD18 transfectants to the MTT dye reduction cytotoxicity assay with various concentrations of Lkt. As expected, Lkt induced cytolysis of the Bo LFA-1 transfectants but not HEK-293 cells (Fig. 2). Interestingly, Lkt induced cytolysis of the Bo CD18 transfectants in a concentration-dependent manner but did not induce cytolysis of the Bo CD11a transfectants (Fig. 2), clearly indicating that Lkt-induced cytolysis is mediated by CD18 and not by CD11a.


Figure 2
View larger version (36K):
[in this window]
[in a new window]

  		FIG. 2. M. haemolytica Lkt induces cytolysis of transfectants expressing monomeric bovine CD18 and heterodimeric LFA-1 but not monomeric CD11a. The bovine transfectants (Bo CD11a, Bo CD18, and Bo LFA-1) and parent cells (HEK-293) were incubated with Lkt, and the percentage of cytolysis was evaluated by MTT dye reduction assay. Results shown are the means of three independent experiments. Error bars indicate standard deviations of the means (*, P < 0.003).

 
Anti-LFA-1 and anti-Lkt MAbs inhibit Lkt-induced cytolysis of transfectants expressing bovine CD18 and LFA-1. Preincubation of Lkt with MM601 greatly inhibited Lkt-induced cytolysis of the Bo CD18 and Bo LFA-1 transfectants (~80%), whereas the isotype-matched control MAb 8G12 had no effect on inhibiting cytolysis, indicating that cytolysis of both Bo CD18 and of Bo LFA-1 was in fact induced by M. haemolytica Lkt (Fig. 3). Likewise, preincubation of transfectants with anti-CD18 MAb inhibited the cytolysis of the Bo CD18 and Bo LFA-1 transfectants (~40%), whereas the isotype-matched control MAb (8G12) did not inhibit the cytolysis (Fig. 4), suggesting that Lkt-induced cytolysis is mediated by CD18. However, anti-CD11a MAb (HUH73A) also inhibited the cytolysis of the Bo LFA-1 transfectant (~50%). In our earlier studies we also observed inhibition of Lkt-induced cytolysis of bovine alveolar macrophages, polymorphonuclear leukocytes, and ovine LFA-1 transfectants by using anti-CD18 and anti-CD11a MAbs (17, 23). However, it should be emphasized here that in the cytotoxicity assay, only the monomeric bovine CD18 transfectants and the heterodimeric LFA-1 transfectants were specifically lysed by Lkt in a concentration-dependent manner (Fig. 2). The Bo CD11a transfectants, like the parent HEK-293 cells, were not lysed by Lkt. Furthermore, the percentages of cytolysis of the monomeric bovine CD18 transfectants and the heterodimeric LFA-1 transfectants were not significantly different from each other. These results clearly indicate that it is the CD18, but not the CD11a, subunit of LFA-1 that serves as a functional receptor for Lkt. The inhibition of Lkt-induced cytolysis by anti-CD18 MAb is very likely due to the direct inhibition of interaction of CD18 with Lkt. However, the inhibition of cytolysis by the anti-CD11a MAb is very likely due to this MAb binding to CD11a and sterically hindering the interaction of CD18 with Lkt. The failure of Lkt to lyse the Bo CD11a transfectants or induce intracellular [Ca2+] elevation in these cells (discussed below) strongly validates this conclusion. Several examples of inhibition of binding of a ligand to its receptor by a MAb binding to a closely juxtaposed molecule have been reported in the literature (25, 30, 33, 39).


Figure 3
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 3. Preincubation of Lkt with Lkt-neutralizing MAb inhibits Lkt-induced cytolysis of transfectants expressing monomeric bovine CD18 or heterodimeric LFA-1. Lkt was preincubated with the Lkt-neutralizing MAb MM601 or isotype-matched control MAb 8G12 before incubation with the bovine transfectants, and the cytotoxicity assay was performed. Results shown are the means of three independent experiments. Error bars indicate standard deviations of the means (*, P < 0.0001).

 

Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		FIG. 4. Anti-LFA-1 MAbs inhibit Lkt-induced cytolysis of transfectants expressing bovine monomeric CD18 or heterodimeric LFA-1. The bovine transfectants were preincubated with anti-bovine CD18 MAb BAQ30A (for Bo CD18 and Bo LFA-1) or anti-human CD11a MAb HUH73A (for Bo LFA-1) or the isotype-matched control MAb 8G12 (for Bo CD18 and Bo LFA-1) before incubation with Lkt, and the cytotoxicity assay was performed. The percentage of inhibition of cytolysis was calculated as described in Materials and Methods. Results shown are the means of three independent experiments. Error bars indicate standard deviations of the means (*, P < 0.0002).

 
Lkt induces intracellular [Ca2+] elevation in the bovine CD18 and LFA-1 transfectants. Lkt induces intracellular [Ca2+] elevation in bovine neutrophils and alveolar macrophages by the influx of extracellular Ca2+ through voltage-gated channels (16, 32). Lkt-induced intracellular [Ca2+] elevation has also been observed by us, using bovine and ovine LFA-1 transductants and transfectants (10, 17, 23). Furthermore, Lkt binding to nonruminant leukocytes does not lead to the elevation of intracellular [Ca2+] (17). The Ca2+ ion is an intracellular second messenger involved in a diverse array of Lkt-induced biological effects and is widely used as an index of intracellular signaling. Hence, it was of interest to determine whether Lkt induces intracellular [Ca2+] elevation in the Bo CD11a and Bo CD18 transfectants. As expected, Lkt induced intracellular [Ca2+] elevation in the Bo LFA-1 transfectants (96%) but not in HEK-293 cells (Fig. 5). More importantly, Lkt induced intracellular [Ca2+] elevation in the Bo CD18 transfectants (88%) but not in the Bo CD11a transfectants (Fig. 5). As little as 5 Lkt units was sufficient to stimulate the elevation of intracellular [Ca2+] in the Bo CD18 and Bo LFA-1 transfectants, whereas even 640 units of Lkt did not induce elevation of intracellular [Ca2+] in the Bo CD11a transfectants and HEK-293 cells (data not shown). Furthermore, the Lkt-induced intracellular [Ca2+] elevations in monomeric bovine CD18 transfectants and in heterodimeric LFA-1 transfectants were not significantly different from each other. Incubation of all three bovine transfectants and HEK-293 cells with culture supernatant from an Lkt mutant strain of M. haemolytica (29) did not result in intracellular [Ca2+] elevation, indicating that the influx of [Ca2+] is specifically mediated by Lkt (Fig. 5). These results confirm that Lkt-induced cytolysis is mediated by Bo CD18 and that Bo CD11a has no role in this process.


Figure 5
View larger version (44K):
[in this window]
[in a new window]

  		FIG. 5. M. haemolytica Lkt induces intracellular [Ca2+] elevation in transfectants expressing monomeric bovine CD18 and heterodimeric LFA-1 but not monomeric CD11a. The bovine transfectants Bo CD11a (panel B), Bo CD18 (panel C), Bo LFA-1 (panel D), and the parent cells (HEK-293, panel A) were incubated with fluorescent calcium indicator (Fluo-4-AM) and exposed to 10 U of Lkt, and intracellular [Ca2+] elevation was analyzed by flow cytometry. Results of one representative experiment out of three are shown.

 
Collectively, the results of this study, along with our previous finding that Lkt induces tyrosine phosphorylation of the cytosolic domain of CD18 but not CD11a (18), now allow us to confidently propose that bovine CD18, the ß subunit of ß2-integrins, is the only functional receptor for M. haemolytica Lkt. Identification of bovine CD18 subunit as the functional receptor for Lkt should facilitate the development of effective control measures to combat pneumonia in ruminants caused by M. haemolytica.


   ACKNOWLEDGMENTS
 
This research was supported by funds from the Foundation for North American Wild Sheep and its eastern Idaho, Oregon, and Washington chapters.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040. Phone: (509) 335-4572. Fax: (509) 335-8529. E-mail: ssrikumaran{at}vetmed.wsu.edu Back

{triangledown} Published ahead of print on 13 August 2007. Back

Editor: R. P. Morrison


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
1. Ambagala, T. C., A. P. Ambagala, and S. Srikumaran. 1999. The leukotoxin of Pasteurella haemolytica binds to beta(2) integrins on bovine leukocytes. FEMS Microbiol. Lett. 179:161-167.[Medline]
2. Baluyut, C. S., R. R. Simonson, W. J. Bemrick, and S. K. Maheswaran. 1981. Interaction of Pasteurella haemolytica with bovine neutrophils: identification and partial characterization of a cytotoxin. Am. J. Vet. Res. 42:1920-1926.[Medline]
3. Bowland, S. L., and P. E. Shewen. 2000. Bovine respiratory disease: commercial vaccines currently available in Canada. Can. Vet. J. 41:33-48.[Medline]
4. Clinkenbeard, K. D., D. A. Mosier, and A. W. Confer. 1989. Effects of Pasteurella haemolytica leukotoxin on isolated bovine neutrophils. Toxicon 27:797-804.[Medline]
5. Confer, A. W., R. J. Panciera, K. D. Clinkenbeard, and D. A. Mosier. 1990. Molecular aspects of virulence of Pasteurella haemolytica. Can. J. Vet. Res. 54:S48-S52.[Medline]
6. Curtis, C. R., M. E. White, and H. N. Erb. 1989. Effect of calfhood morbidity on long term survival in New York Holstein herds. Prev. Vet. Med. 7:173-186.[CrossRef]
7. Dassanayake, R. P., S. Shanthalingam, W. C. Davis, and S. Srikumaran. 2007. Mannheimia haemolytica leukotoxin-induced cytolysis of ovine (Ovis aries) leukocytes is mediated by CD18, the ß subunit of ß2-integrins. Microb. Pathog. 42:167-173.[CrossRef][Medline]
8. Deshpande, M. S., T. C. Ambagala, A. P. N. Ambagala, M. E. Kehrli, Jr., and S. Srikumaran. 2002. Bovine CD18 is necessary and sufficient to mediate Mannheimia (Pasteurella) haemolytica leukotoxin-induced cytolysis. Infect. Immun. 70:5058-5068.[Abstract/Free Full Text]
9. Dileepan, T., M. S. Kannan, B. Walcheck, P. Thumbikat, and S. K. Maheswaran. 2005. Mapping of the binding site for Mannheimia haemolytica leukotoxin within bovine CD18. Infect. Immun. 73:5233-5237.[Abstract/Free Full Text]
10. Dileepan, T., P. Thumbikat, B. Walcheck, M. S. Kannan, and S. K. Maheswaran. 2005. Recombinant expression of bovine LFA-1 and characterization of its role as a receptor for Mannheimia haemolytica leukotoxin. Microb. Pathog. 38:249-257.[CrossRef][Medline]
11. Gahmberg, C. G., L. Valmu, S. Fagerholm, P. Kotovuori, E. Ihanus, L. Tian, and T. Pessa-Morokawa. 1998. Leukocyte integrins and inflammation. Cell. Mol. Life Sci. 54:549-555.[CrossRef][Medline]
12. Gentry, M. J., and S. Srikumaran. 1991. Neutralizing monoclonal antibodies to Pasteurella haemolytica leukotoxin affinity-purify the toxin from crude culture supernatants. Microb. Pathog. 10:411-417.[CrossRef][Medline]
13. Griffin, D. 1997. Economic impact associated with respiratory disease in beef cattle. Vet. Clin. North Am. Food Anim. Pract. 13:367-377.[Medline]
14. Highlander, S. K., N. D. Fedorova, D. M. Dusek, R. Panciera, L. E. Alvarez, and C. Renehart. 2000. Inactivation of Pasteurella (Mannheimia) haemolytica leukotoxin causes partial attenuation of virulence in a calf challenge model. Infect. Immun. 68:3916-3922.[Abstract/Free Full Text]
15. Hodgson, J. C., J. M. Moon, M. Quirie, and W. Donachie. 2003. Association of LPS chemotype of Mannheimia (Pasteurella) haemolytica A1 with disease virulence in a model of ovine pneumonic pasteurellosis. J. Endotoxin Res. 9:25-32.[Medline]
16. Hsuan, S. L., M. S. Kannan, S. Jeyaseelan, Y. S. Prakash, G. C. Sieck, and S. K. Maheswaran. 1998. Pasteurella haemolytica A1-derived leukotoxin and endotoxin induce intracellular calcium elevation in bovine alveolar macrophages by different signaling pathways. Infect. Immun. 66:2836-2844.[Abstract/Free Full Text]
17. Jeyaseelan, S., S. L. Hsuan, M. S. Kannan, B. Walcheck, J. F. Wang, M. E. Kehrli, E. T. Lally, G. C. Sieck, and S. K. Maheswaran. 2000. Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes. Infect. Immun. 68:72-79.[Abstract/Free Full Text]
18. Jeyaseelan, S., M. S. Kannan, R. E. Briggs, P. Thumbikat, and S. K. Maheswaran. 2001. Mannheimia haemolytica leukotoxin activates a nonreceptor tyrosine kinase signaling cascade in bovine leukocytes, which induces biological effects. Infect. Immun. 69:6131-6139.[Abstract/Free Full Text]
19. Jeyaseelan, S., S. Sreevatsan, and S. K. Maheswaran. 2002. Role of Mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis. Anim. Health Res. Rev. 3:69-82.[CrossRef][Medline]
20. Kehrli, M. E., Jr., M. R. Ackermann, D. E. Shuster, M. J. van der Maaten, F. C. Schmalstieg, D. C. Anderson, and B. J. Hughes. 1992. Bovine leukocyte adhesion deficiency. ß2 integrin deficiency in young Holstein cattle. Am. J. Pathol. 140:1489-1492.[Medline]
21. Klucas, C. A., and G. A. Anderson. 1988. Bovine respiratory syncytial virus-specific monoclonal antibodies. Vet. Immunol. Immunopathol. 18:307-315.[CrossRef][Medline]
22. Larson, R. S., M. L. Hihbs, and T. A. Springer. 1990. The leukocyte integrin LFA-I reconstituted by cDNA transfection in a nonhematopoietic cell line is functionally active and not transiently regulated. Cell Regul. 1:359-367.[Medline]
23. Lawrence, P. K., R. P. Dassanayake, D. P. Knowles, and S. Srikumaran. 6 May 2007, posting date. Transfection of non-susceptible cells with Ovis aries recombinant lymphocyte function-associated antigen 1 renders susceptibility to Mannheimia haemolytica leukotoxin. Vet. Microbiol. [Epub ahead of print.] doi:10.1016/j.vetmic.2007.05.006.
24. Li, J., K. D. Clinkenbeard, and J. W. Ritchey. 1999. Bovine CD18 identified as a species specific receptor for Pasteurella haemolytica leukotoxin. Vet. Microbiol. 67:91-97.[CrossRef][Medline]
25. List, K., G. Hoyer-Hansen, F. Ronne, K. Dano, and N. Behrendt. 1999. Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology. J. Immunol. Methods 222:125-133.[CrossRef][Medline]
26. Liu, W., K. A. Brayton, W. C. Davis, K. Mansfield, J. Lagerquist, W. J. Foreyt, and S. Srikumaran. 2007. Mannheimia (Pasteurella) haemolytica leukotoxin utilizes CD18 as its receptor on bighorn sheep leukocytes. J. Wildl. Dis. 43:75-81.[Abstract/Free Full Text]
27. Luscinskas, F. W., A. F. Brock, M. A. Arnaout, and M. A. Gimbrone, Jr. 1989. Endothelial-leukocyte adhesion molecule-1-dependent and leukocyte (CD11/CD18)-dependent mechanisms contribute to polymorphonuclear leukocyte adhesion to cytokine-activated human vascular endothelium. J. Immunol. 142:2257-2263.[Abstract]
28. Marlin, S. D., C. C. Morton, D. C. Anderson, and T. A. Springer. 1986. LFA-1 immunodeficiency disease. Definition of the genetic defect and chromosomal mapping of alpha and beta subunits of the lymphocyte function associated antigen 1 (LFA-1) by complementation in hybrid cells. J. Exp. Med. 164:855-867.[Abstract/Free Full Text]
29. Murphy, G. L., L. C. Whitworth, K. D. Clinkenbeard, and P. A. Clinkenbeard. 1995. Hemolytic activity of the Pasteurella haemolytica leukotoxin. Infect. Immun. 63:3209-3212.[Abstract]
30. Nason, E. L., J. D. Wetzel, S. K. Mukherjee, E. S. Barton, B. V. Prasad, and T. S. Dermody. 2001. A monoclonal antibody specific for reovirus outer-capsid protein {sigma}3 inhibits {sigma}1-mediated hemagglutination by steric hindrance. J. Virol. 75:6625-6634.[Abstract/Free Full Text]
31. Noti, J. D., A. K. Johnson, and J. D. Dillon. 2000. Structural and functional characterization of the leukocyte integrin gene CD11d. Essential role of Sp1 and Sp3. J. Biol. Chem. 275:8959-8969.[Abstract/Free Full Text]
32. Ortiz-Carranza, O., and C. J. Czuprynski. 1992. Activation of bovine neutrophils by Pasteurella haemolytica leukotoxin is calcium dependent. J. Leukoc. Biol. 52:558-564.[Abstract]
33. Petersen, H. H., M. Hansen, S. L. Schousboe, and P. A. Andreasen. 2001. Localization of epitopes for monoclonal antibodies to urokinase-type plasminogen activator: relationship between epitope localization and effects of antibodies on molecular interactions of the enzyme. Eur. J. Biochem. 268:4430-4439.[Medline]
34. Shuster, D. E., B. T. Bosworth, and M. E. Kehrli, Jr. 1992. Sequence of the bovine CD18-encoding cDNA: comparison with the human and murine glycoproteins. Gene 14:267-271.
35. Solovjov, D. A., E. Pluskota, and E. F. Plow. 2005. Distinct roles for the {alpha} and ß subunits in the functions of integrin {alpha}Mß2. J. Biol. Chem. 280:1336-1345.[Abstract/Free Full Text]
36. Sutherland, A. D. 1985. Effects of Pasteurella haemolytica cytotoxin on ovine peripheral blood leucocytes and lymphocytes obtained from gastric lymph. Vet. Microbiol. 10:431-438.[CrossRef][Medline]
37. Tatum, F. M., R. E. Briggs, S. S. Sreevatsan, E. S. Zehr, S. L. Hsuan, L. O. Whiteley, T. R. Ames, and S. K. Maheswaran. 1998. Construction of an isogenic leukotoxin deletion mutant of Pasteurella haemolytica serotype 1: characterization and virulence. Microb. Pathog. 24:37-46.[CrossRef][Medline]
38. Thumbikat, P., T. Dileepan, M. S. Kannan, and S. K. Maheswaran. 2005. Characterization of Mannheimia (Pasteurella) haemolytica leukotoxin interaction with bovine alveolar macrophage beta2 integrins. Vet. Res. 36:771-786.[CrossRef][Medline]
39. van Oss, C. J. 1997. Kinetics and energetics of specific intermolecular interactions. J. Mol. Recognit. 10:203-216.[CrossRef][Medline]
40. Wang, J. F., I. R. Kieba, J. Korostoff, T. L. Guo, N. Yamaguchi, H. Rozmiarek, P. C. Billings, B. J. Shenker, and E. T. Lally. 1998. Molecular and biochemical mechanisms of Pasteurella haemolytica leukotoxin-induced cell death. Microb. Pathog. 25:317-331.[CrossRef][Medline]
41. Whiteley, L. O., S. K. Maheswaran, D. J. Weiss, and M. S. Kannan. 1992. Pasteurella haemolytica A1 and bovine respiratory disease: pathogenesis. J. Vet. Int. Med. 6:11-22.
42. Xiong, Y. M., and L. Zhang. 2001. Structure-function of the putative I-domain within the integrin ß2 subunit. J. Biol. Chem. 276:19340-19349.[Abstract/Free Full Text]

Infection and Immunity, October 2007, p. 5004-5010, Vol. 75, No. 10
0019-9567/07/$08.00+0     doi:10.1128/IAI.00808-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
IAI.00808-07v1
75/10/5004    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dassanayake, R. P.
Right arrow Articles by Srikumaran, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dassanayake, R. P.
Right arrow Articles by Srikumaran, S.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. J. Virol. Eukaryot. Cell
Microbiol. Mol. Biol. Rev. Clin. Vaccine Immunol. All ASM Journals