Monomeric Expression of Bovine β2-integrin Subunits Reveals their Role in
Mannheimia haemolytica Leukotoxin-induced Biological Effects
Rohana P. Dassanayake1, Samuel K. Maheswaran2 and Subramaniam Srikumaran1
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA.
Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, MN 55108, USA.
ABSTRACT
*
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
US and elsewhere. This bacterium produces several virulence determinants out of which, leukotoxin (Lkt) and
lipopolysaccharide are the major determinants that largely contribute to the pathogenesis of pneumonia. Lkt is a member
of the repeats-in-toxin (RTX) family of pore-forming cytolysins that shows cell type as well as species specificity.
Previously, we and others have shown that the cytotoxic effect of Lkt on bovine and ovine leukocytes is mediated by Lkt-β2
integrin interactions. β2 integrins are leukocyte-specific integrins, and are expressed on the cell surface as heterodimeric
glycoproteins composed of the α subunit CD11 and the β subunit CD18. These subunit associations give rise to 4 different
β2 integrins. 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 β2 integrin(s) is the receptor for Lkt. Indeed, in our
previous studies, recombinant expression of bovine or ovine CD18 in an Lkt non-susceptible cell line rendered it
susceptible to Lkt-induced cytolysis indicating that CD18 is necessary and sufficient to mediate Lkt-induced cytolysis of
target cells. 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. In addition, antibodies specific for CD18 or CD11a were equally effective in inhibiting
signaling events leading to elevation of intracellular [Ca2+]i, tyrosine phosphorylation of the cytosolic domain of CD18, and
cytolysis of bovine leukocytes. 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. We took advantage of the ability of HEK-293 cell line to express monomeric CD11a and CD18, as well as its
non-susceptibility 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. Therefore, the objectives of this study were to:
(i) determine if binding of Lkt to CD18 alone has any functional consequence; (ii) determine if CD18 dimerization with
CD11a is required for responsiveness to Lkt-induced biological effects; (iii) determine if binding of Lkt to CD11a has any
functional consequence; and (iv) determine which one of these subunits serves as the functional receptor for M.
haemolytica Lkt. Here, we present data which demonstrates unequivocally that bovine CD18 is the functional receptor for
M. haemolytica Lkt.
RESULTS
101
102
HEK-293
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
100
103
104
(D)115
110
HEK-293
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
Bo CD18
Count
Bo CD11a
(C)115
110
Bo LFA1
(CD11a)
101
FL1-H
102
103
104
100
FL1-H
101
102
103
104
100
101
102
103
(A) 85
(B) 100
95
(C) 115
110
80
90
75
85
70
80
65
75
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
65
Count
Count
50
45
Bo CD11a - Lkt
40
55
Bo CD18 - Lkt
50
45
35
40
30
35
30
25
25
20
20
15
15
10
10
5
5
0
0
10
HEK-293 - Lkt
60
10
1
% Cytolysis
0
160
10
2
FL1-H
10
3
10
4
10 0
10 1
10 2
FL1-H
10 3
10
4
80
-10
40
20
10
5
Toxin units/ml
Fig. 3. 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 % cytolysis was evaluated by MTT dye reduction cytotoxicity
assay. Transfectants expressing monomeric bovine CD18 and heterodimeric LFA-1, but not
monomeric CD11a, or the parent cells, were efficiently lysed by leukotoxin (Lkt) in a
concentration-dependent manner. Results shown are the means of three independent
experiments. The error bars indicate standard deviations of the means (* P <0.003).
100
(A)
*
*
Bo CD18 – MM601
80
Bo LFA-1 – MM601
60
Bo CD18/
Bo LFA-1 – 8G12
40
20
*
50
*
*
Bo CD18
Bo LFA-1
40
Bo CD18 – BAQ30A
Bo LFA-1 – BAQ30A
Bo LFA-1 – HUH73A
Bo CD18/
Bo LFA-1 – 8G12
30
20
10
0
0
Bo CD18
-20
(B) 60
Bo LFA-1
Bo CD18/
Bo LFA-1
MAbs (10 μg/ml)
-10
Bo LFA-1
MAbs (50 μg/ml)
Bo CD18/
Bo LFA-1
Fig. 4. Anti-Lkt and anti-LFA-1 MAbs inhibit Lkt-induced cytolysis of transfectants
expressing monomeric bovine CD18 or heterodimeric LFA-1.
In two separate experiments, leukotoxin (Lkt) was pre-incubated with Lkt-neutralizing MAb
MM-601 (Panel A), and bovine transfectants were preincubated with anti-CD18 MAb BAQ30A
or anti-CD11a MAb HUH73A (Panel B), and the cytotoxicity assay was conducted as in Fig. 3.
The MAb 8G12 was used as isotype-matched control in both experiments. The anti-Lkt MAb
(MM601), and anti-CD18 MAb (BAQ30A) inhibited the Lkt-induced cytolysis of Bo CD18 and
Bo LFA-1 transfectants indicating that the cytolysis was induced by Lkt and mediated by
CD18. Inhibition of cytolysis of Bo LFA-1 transfectants by anti-CD11a MAb is very likely due
to steric hinderance because Lkt does not lyse Bo CD11a transfectants expressing CD11a
alone (Fig. 3). Results shown are the means of three independent experiments. The error
bars indicate standard deviations of the means (* P <0.002).
(B)
(C)
(D)
10 0
Fig 5. M. haemolytica Lkt induces intracellular [Ca2+]i elevation in transfectants
expressing monomeric bovine CD18 and heterodimeric LFA-1, but not monomeric
CD11a.
The parent cells (HEK-293, Panel A), bovine transfectants Bo CD11a (Panel B), Bo CD18
(Panel C), and Bo LFA-1 (Panel D) were incubated with fluorescent calcium indicator (Fluo-4AM) and exposed to 10U of leukotoxin (Lkt). Intracellular [Ca2+]i elevation was analyzed by
flow cytometry. Lkt from M. haemolytica wild type (Wt), but not culture supernatant from Lkt
deletion mutant (Mt), induced intracellular [Ca2+]i elevation in transfectants expressing
monomeric bovine CD18 or heterodimeric LFA-1. There was no intracellular [Ca2+]i elevation
in transfectants expressing monomeric CD11a, or the parent cells. These observations
revealed that intracellular [Ca2+]i elevation, which is considered as an indication of Lktreceptor interaction, was mediated only by bovine CD18, confirming CD18 as the functional
receptor for Lkt. Results of one representative experiment out of three are shown.
CONCLUSIONS
HEK-293 - Lkt
Count
HEK-293 - Lkt
0
10
104
Transfectants stably expressing monomeric bovine CD11a (Bo CD11a), or monomeric bovine CD18 (Bo CD18),
or heterodimeric bovine CD11a/CD18 (Bo LFA-1) were developed by transfection of the non-hematopoietic
human cell-line HEK-293 with plasmids carrying cDNA for bovine CD11a or CD18 or co-transfection with both
plasmids, respectively. The transfectants Bo CD11a [Panel A], Bo CD18 [Panel B] and Bo LFA-1 [Panels C and
D]) and parent cells (HEK-293 [Panels A-D]) were incubated with either anti-bovine CD11a (Panels A and C) or
anti-bovine CD18 (Panels B and D) MAbs followed by FITC-conjugated goat anti-mouse Ig antibodies. Flow
cytometric analysis confirmed the surface expression of bovine CD11a, CD18 and LFA-1 on the respective
transfectants, but not the parent cells. Results of one representative experiment out of three are shown.
55
20
FL1-H
FL1-H
70
30
Bo LFA1
(CD18)
Fig. 1. Cell surface expression of monomeric bovine CD11a, CD18, or heterodimeric LFA-1 by
transfectants.
60
*
HEK-293
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
Count
HEK-293
Count
110
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
100
(B)115
110
*
40
(A)
Count
(A)120
115
*
HEK-293
% Inhibition of cytolysis
INTRODUCTION
*
50
% Inhibition of cytolysis
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 β subunit of bovine β2-integrins on
leukocytes, and the species specificity of Lkt-induced effects is resident in the CD18 subunit, and not in
the α 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+]i, 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 LFA1 utilized by M. haemolytica Lkt as the functional receptor. For this purpose, monomeric bovine CD18,
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+]i elevation following exposure to Lkt,
which is a marker for post-binding 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 β2-integrins is the functional receptor for M. haemolytica Lkt.
Bo LFA-1
Bo CD18
Bo CD11a
*
60
Bo LFA-1 - Lkt
10 1
10 2
10 3
10 4
FL1-H
Fig. 2. M. haemolytica Lkt binds to transfectants expressing monomeric bovine CD11a, CD18 or
heterodimeric LFA-1.
The bovine transfectants [Bo CD11a (Panel A), Bo CD18 (Panel B) and Bo LFA-1 (Panel C)] and parent cells
(HEK-293, Panels A-C) were incubated with leukotoxin (Lkt), fixed with 2% paraformaldehyde, washed, and
incubated with FITC-conjugated Lkt non-neutralizing MAb MM605. Flow cytometric analysis revealed that Lkt
efficiently bound to all three bovine transfectants, but not to the parent cells. Results of one representative
experiment out of three are shown.
In this study, Lkt bound transfectants expressing monomeric CD18, monomeric CD11a, or
heterodimeric CD11a/CD18. However, Lkt-induced cytolysis was observed only with transfectants
expressing monomeric CD18 or CD11a/CD18, but not monomeric CD11a, indicating that Lkt-induced
cytolysis is mediated by CD18 alone. Lkt-neutralizing MAb and anti CD18 MAb inhibited the cytolysis
of transfectants expressing CD18 or CD11a/CD18 confirming that the cytolysis was induced by Lkt,
and mediated by CD18. Intracellular [Ca2+]i elevation, which is an indication of Lkt-receptor
interaction, was observed only in transfectants expressing CD18 or CD11a/CD18. Based on these
findings, we confidently propose that bovine CD18, the β subunit of β2-integrins, is the only functional
receptor for M. haemolytica Lkt. However, the association of CD11a with CD18 in the cytosol greatly
enhances the transport and stable expression of both subunits on the cell surface.
ACKNOWLEDGMENTS
This research was supported by funds from the Foundation for North American Wild Sheep and its Eastern,
Idaho, Oregon, and Washington Chapters.