RTX Toxins Recognize a β2 Integrin on the Surface of Human Target Cells

Actinobacillus actinomycetemcomitans leukotoxin and Escherichia coliα-hemolysin are RTX toxins that kill human immune cells. We have obtained a monoclonal antibody (295) to a cell surface molecule present on toxin-sensitive HL60 cells that can inhibit cytolysis by both RTX toxins. Utilization of this monoclonal antibody for immunoaffinity purification of detergent-solubilized target cell membranes yielded two polypeptide chains of approximate molecular masses of 100 and 170 kDa. Microsequencing of tryptic peptides from the two proteins showed complete homology with CD11a and CD18, the two subunits of the β2 integrin, lymphocyte function-associated antigen 1 (LFA-1). Anti-CD11a and CD18 monoclonal antibodies also inhibited RTX toxin-mediated cytolysis. Direct binding experiments demonstrated the ability of an immobilized RTX to bind LFA-1 heterodimers present in a detergent lysate of human HL60 target cells. Transfection of CD11a and CD18 integrin genes into a cell line (K562) that is not sensitive to either RTX toxin resulted in LFA-1 expressing cells, KL/4, that were sensitive to both toxins. These experiments identify LFA-1 as a cell surface receptor that mediates toxicity of members of this family of pore-forming toxins. Actinobacillus actinomycetemcomitans leukotoxin and Escherichia coliα-hemolysin are RTX toxins that kill human immune cells. We have obtained a monoclonal antibody (295) to a cell surface molecule present on toxin-sensitive HL60 cells that can inhibit cytolysis by both RTX toxins. Utilization of this monoclonal antibody for immunoaffinity purification of detergent-solubilized target cell membranes yielded two polypeptide chains of approximate molecular masses of 100 and 170 kDa. Microsequencing of tryptic peptides from the two proteins showed complete homology with CD11a and CD18, the two subunits of the β2 integrin, lymphocyte function-associated antigen 1 (LFA-1). Anti-CD11a and CD18 monoclonal antibodies also inhibited RTX toxin-mediated cytolysis. Direct binding experiments demonstrated the ability of an immobilized RTX to bind LFA-1 heterodimers present in a detergent lysate of human HL60 target cells. Transfection of CD11a and CD18 integrin genes into a cell line (K562) that is not sensitive to either RTX toxin resulted in LFA-1 expressing cells, KL/4, that were sensitive to both toxins. These experiments identify LFA-1 as a cell surface receptor that mediates toxicity of members of this family of pore-forming toxins. A paradigm emerging from studies of bacterial protein toxins is that target cell recognition is often the initial event of a multistep process ultimately leading to diverse mechanisms of cell death. For example, colicins (1Pattus F. Massotte D. Wilmsen H.U. Lakey J. Tsernoglou D. Tucker A.X. Parker M.W. Experientia. 1990; 46: 180-192PubMed Google Scholar) bind to various outer membrane proteins ofEscherichia coli (2Parker M.W. Tucker A.D. Tsernoglou D. Pattus F. Trends Biochem. Sci. 1990; 15: 126-129Abstract Full Text PDF PubMed Scopus (114) Google Scholar), Bacillus thuringiensisδ-endotoxin recognizes the brush border of insect gut cells (3Van Rie J. Jansens S. Hofte H. Degheele D. Van Mellaert H. Appl. Environ. Microbiol. 1990; 56: 1378-1385Crossref PubMed Google Scholar), diphtheria toxin binds to a heparin-binding epidermal growth factor-like precursor (4Van Rie J. Jansens S. Hofte H. Degheele D. Van Mellaert H. Eur. J. Biochem. 1989; 186: 239-247Crossref PubMed Scopus (280) Google Scholar), and the family of AB5 toxins (e.g. cholera toxin) bind to cell surface ganglioside lipids (5Merritt E.A. Hol W.G. Curr. Opin. Struct. Biol. 1995; 5: 165-171Crossref PubMed Scopus (245) Google Scholar). Actinobacillus actinomycetemcomitans leukotoxin is a member of the RTX toxin family of pore-forming hemolysins/leukotoxins (6Strathdee C.A. Lo R.Y. J. Bacteriol. 1989; 171: 916-928Crossref PubMed Google Scholar, 7Koronakis V. Hughes C. Koronakis E. Mol. Microbiol. 1993; 8: 1163-1175Crossref PubMed Scopus (79) Google Scholar, 8Lo R.Y. Shewen P.E. Strathdee C.A. Greer C.N. Infect. Immun. 1985; 50: 667-671Crossref PubMed Google Scholar, 9Lally E.T. Kieba I.R. Demuth D.R. Rosenbloom J. Golub E.E. Taichman N.S. Gibson C.W. Biochem. Biophys. Res. Commun. 1989; 159: 256-262Crossref PubMed Scopus (74) Google Scholar, 10Lian C.J. Rosendal S. MacInnes J.I. Infect. Immun. 1989; 57: 3377-3382Crossref PubMed Google Scholar, 11Mobley H.L. Chippendale G.R. Swihart K.G. Welch R.A. Infect. Immun. 1991; 59: 2036-2042Crossref PubMed Google Scholar). Leukotoxin kills cells of the lymphocytic and monomyelocytic lineage of man and some higher nonhuman primates. The narrow host range of leukotoxin suggests that the toxin may bind a specific cell surface receptor. However, no target cell receptors for RTX toxins have yet to be identified. Both toxin-sensitive and several toxin-resistant cells (12Simpson D.L. Berthold P. Taichman N.S. Infect. Immun. 1988; 56: 1162-1166Crossref PubMed Google Scholar) are capable of absorbing toxin from culture supernatants, indicating that simple adsorption of the toxin onto a cell membrane is not enough to cause cell death. The ability of these toxins to adhere to various cell surfaces also indicates that classical experiments designed to detect the presence of a receptor such as saturation of binding or inhibition of labeled toxin binding with excess unlabeled toxin will not be effective in detecting cell surface receptors for this family of toxins. We have employed an alternative approach to identify a cell surface molecule that mediates RTX toxin cytotoxicity. Balb/cJ female mice (Jackson Laboratories, Bar Harbor, ME) 12–16 weeks old were immunized intravenously with 2 × 107 HL60 cells, boosted on days 10, 20, and 30 with an equal number of cells and allowed to rest for 60 days. 3 days before fusion, the animals again received 2 × 107 HL60 cells intravenously. On the day of fusion, spleens were removed aseptically, and a single cell suspension was prepared with a loose fitting tissue homogenizer. The cells were washed once in Dulbecco's minimal essential medium, and the erythrocytes were lysed with 0.17m ammonium chloride-Tris buffer. Spleen cells recovered in this manner had a viability of >95% as assessed by trypan blue exclusion. Sp2/0-Agl4 myeloma cells were mixed with spleen cells (1:10) and centrifuged. A 1-ml portion of 30% polyethylene glycol solution (PEG 1000; J. T. Baker Chemical Co., Phillipsburg, NJ) was slowly added to the cell pellet. The pellet was gently stirred, allowed to set for 1 min, and then dispersed by the addition of 50 ml of Dulbecco's minimal essential medium. After centrifugation, the cells were suspended in 30 ml of Kennett's HY medium (4.5 g/liter Dulbecco's minimal essential medium with high glucose, glutamine, 10% NCTC 109, 20% fetal bovine serum, 1% 0.15 mg/ml oxaloacetate, 0.05 mg/ml pyruvate, and 0.2 unit/ml bovine insulin) and 1% 5 mmhypoxanthine and 0.8 mm thymidine), and 0.1-ml portions were placed in 96-well tissue culture plates. The next day, an additional 0.1 ml of medium containing aminopterin (0.04 μm) was added to each well. Cells were fed every 3–4 days by drawing off 0.1 ml of spent medium. Clones were visible 7–9 days after the fusion, and when they covered approximately one-half of the bottom of the well, they were screened for their ability to inhibit RTX-mediated cytotoxicitv in the biological assay (13Lally E.T. Golub E.E. Kieba I.R. J. Biol. Chem. 1994; 269: 31289-31295Abstract Full Text PDF PubMed Google Scholar, 14Brogan J.M. Lally E.T. Poulsen K. Kilian M. Demuth D.R. Infect. Immun. 1994; 62: 501-508Crossref PubMed Google Scholar). The anti-integrin mAbs 1The abbreviations used are: mAb, monoclonal antibody; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; LFA-1, lymphocyte function-associated antigen 1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; GADPH, glyceraldehyde-3-phosphate dehydrogenase; bp, base pair(s); FACS, fluorescence-activated cell sorter; ICAM, intercellular adhesion molecule. used in the study were: TS1/18 (15Sanchez-Madrid F. Krensky A.M. Ware C.F. Robbins E. Strominger J.L. Burakoff S.J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7489-7493Crossref PubMed Scopus (587) Google Scholar), KIM127 (16Robinson M.K. Andrew D. Rosen H. Brown D. Ortlepp S. Stephens P.X. Butcher E.C. J. Immunol. 1992; 148: 1080-1085PubMed Google Scholar), KIM185 (16Robinson M.K. Andrew D. Rosen H. Brown D. Ortlepp S. Stephens P.X. Butcher E.C. J. Immunol. 1992; 148: 1080-1085PubMed Google Scholar), (IgG1,κ) antibodies that bind to CD18; 38 (17Dransfield I. Hogg N. EMBO J. 1989; 8: 3759-3765Crossref PubMed Scopus (266) Google Scholar) (IgG2a,κ) and 25.3.1 (17Dransfield I. Hogg N. EMBO J. 1989; 8: 3759-3765Crossref PubMed Scopus (266) Google Scholar) and TS1/22 (15Sanchez-Madrid F. Krensky A.M. Ware C.F. Robbins E. Strominger J.L. Burakoff S.J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7489-7493Crossref PubMed Scopus (587) Google Scholar) (IgG1,κ) antibodies that bind to CD11a; 44 (18Third International Workshop and Conference on Human Leucocyte Differentiation Antigens Leucocyte Typing III: White Cell Differentiation Antigens. Oxford University Press, Oxford1987Google Scholar), an (IgG1,κ) antibody that binds to CD11b; and 3.9 (19Hogg N. Takacs L. Palmer D.G. Selvendran Y. Allen C. Eur. J. Immunol. 1986; 16: 240-248Crossref PubMed Scopus (114) Google Scholar) an (IgG1,κ) antibody that binds to CD11c. Leukotoxic activity of recombinant ltxA and hylA gene products was determined essentially as described previously (13Lally E.T. Golub E.E. Kieba I.R. J. Biol. Chem. 1994; 269: 31289-31295Abstract Full Text PDF PubMed Google Scholar). Briefly, E. coli containingltxC and either ltxA or hylA in the pOTSNco12 plasmid were grown to late log phase, induced, and sonicated. The bacterial sonicate was incubated with target cells at 37 °C for 45 min. RTX-sensitive HL60 cells were cultured in RPMI 1640 and used as positive controls, whereas negative controls consisted of incubating target cells with: (i) tissue culture medium, (ii) sonicates from uninduced bacteria, or (iii) sonicates from induced bacteria that contained the pOTSNco12 plasmid without an insert. The cells were placed on ice, 100 μl of trypan blue (0.4%) were added, and surviving cells were counted in a hemocytometer. At least four fields were counted in triplicate and averaged for each dilution assayed. The percentage of lysis was calculated by dividing the number of surviving cells by the number of cells in the negative controls and subtracting from 100. Prior to incubation with anti-integrin antibodies, HL60, KL/4, and K562 cells were washed twice in cold 1% BSA PBS and incubated with mouse IgG myeloma protein (10 μg; 30 min on ice) that was the same isotype as the anti-integrin antibody. Immunoglobulin bound during this incubation occurs the receptor. After again in cold 1% BSA PBS, the cells were incubated with an anti-integrin antibody for min, and incubated for an additional min with IgG The of a of cells, mouse IgG myeloma and IgG the to The of were then on the to the of anti-integrin antibody bound on the cells. After cells were in and at °C cells were on a HL60 cells were × min, and washed with PBS, and the pellet was in lysis mm mm CHAPS, 10% 1 mm 5 5 The suspension was on for 30 min and × min, and the was used for protein The was with ml of The was by min, and washed with ml of (10 mm 1 20, protein was with 100 mm 100 mm The protein was mm and by The proteins were on were μm) and at °C to the were and allowed to for at to and 0.1 was added to the to proteins were in 5 × containing and at 37 °C for min. After proteins were to a Prior to membrane was in for and then in with 10% for 5 min. After the membrane was with for 5 min. The membrane was with and proteins of 100 and 170 were from the membrane and with The tryptic were by HPLC, and peptides from each protein were for A. Lally E.T. Biophys. 1995; PubMed Scopus Google Scholar) was in PBS and incubated (10 of toxin) in a with at The were washed with PBS and incubated with 1 mg/ml BSA in PBS for 1 to the (10 of of cells were incubated with the for at °C in After the were washed with containing by a in mm protein was from the gel and on were to a and the membrane was in PBS, and incubated with antibodies for at The membranes were washed with and incubated with a in PBS, for 1 at The were washed with and K562 S. Stephens P.E. Hogg N. M.K. Eur. J. Immunol. 1995; PubMed Scopus Google Scholar) were by the CD11a and CD18 genes into a of the P.E. Res. 1989; PubMed Scopus Google Scholar) a After the of the integrin genes L. Springer T. J. Cell Biol. 1989; PubMed Scopus Google Scholar, J. EMBO J. PubMed Scopus Google Scholar) was by the in is present in both and cells, was used as a gene S. S. T. C. The Scholar). was prepared from and K562 cell the purification by to the was a and the The for were: CD18, CD18, GADPH, GADPH, The were to °C for min by 30 at °C for °C for and °C for and an of min at was membrane to the L. Springer T. J. Cell Biol. 1989; PubMed Scopus Google Scholar, J. EMBO J. PubMed Scopus Google Scholar) were labeled the and The for were: CD18, experiments were designed to identify a monoclonal antibody that for a cell surface molecule on target cells and is capable of RTX-mediated cytotoxicity. Balb/cJ mice were immunized with HL60 cells the from immunized mice were removed and with a mouse myeloma cell culture were screened for their ability to inhibit HL60 by two RTX E. coli actinomycetemcomitans was to be an of the activity of both toxins cell demonstrated that the antigen on the cell surface of human and 1 identify the HL60 cell membrane were with CHAPS, a J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar), and then with of the two proteins of molecular masses of 100 and 170 had bound to the of these was when the same HL60 lysate was an with a myeloma protein proteins from a were to and onto After with proteins of were from the and to were by and by tryptic peptides from the protein were and showed complete homology with from whereas peptides from the were and to be to of CD18 The CD11a and CD18 the and of a β2 integrin, of tryptic peptides from and proteins present in the from a was determined by not was a recovered with from the immunoaffinity were to and to and 170 were from the and with and peptides for were then used to the protein and were was determined by not was a recovered with in a from the immunoaffinity were to and to and 170 were from the and with and peptides for were then used to the protein and were identified. of LFA-1 as the molecule that is by to the that and monoclonal antibodies also inhibit RTX-mediated cytotoxicity. A of monoclonal antibodies was screened to identify RTX toxin-mediated an and antibodies and were capable of RTX cytolysis. The that the were to receptor binding was by incubating cells with an (IgG1,κ) inhibition of RTX was to anti-integrin antibodies binding the receptor and not to the β2 integrin, then be effective target cell as the anti-integrin was not the of LFA-1 by antibodies and that were used in a of TS1/22 3 an antibody the of CD11a C. Springer T.A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), inhibited an that the also on mechanisms for inhibition of RTX-mediated by integrin monoclonal A is The monoclonal antibody then with binding of the toxin to the same integrin the toxin not bind to β2 the of antibody and integrin in the cell to toxin binding at a this a of experiments the ability of RTX toxins to bind to β2 was A. actinomycetemcomitans leukotoxin was onto and additional protein binding was by incubating the with The toxin were then incubated with a lysate from HL60 cells. protein was from the on onto and incubated with either 25.3.1 or KIM185 The that CD11a and CD18 are in the from RTX toxin with BSA showed no of binding of either integrin 2 when of A. actinomycetemcomitans leukotoxin was added to the the ability of the RTX toxin to bind either of the LFA-1 was and binding determined the ability of to LFA-1 toxin of with (10 integrin binding to and inhibition of binding was when the were incubated with an of an isotype 3 and A cell line was employed to the of CD18 and CD11a genes in RTX toxin-mediated cytotoxicity. a human cell line that not CD11a or CD18 was with both CD11a and CD18 in a single of the cell KL/4, was to both CD11a and CD18 genes of the and products for CD11a and CD18 genes in cells. for CD11a or CD18 was in cells and Both cell of the 3 and The and products from to of CD11a and CD18 of cells demonstrated cell surface of CD11a and CD18 gene whereas no of LFA-1 was on K562 cells. of the two proteins indicating that of the two proteins is the to of either or chains that LFA-1 is the on cells. of the LFA-1 on cells described previously S. Stephens P.E. Hogg N. M.K. Eur. J. Immunol. 1995; PubMed Scopus Google Scholar). by cells to be in a of as determined by their to bind either or antibodies that of β2 integrin heterodimers in K562 number and and and number of K562 cells and LFA-1 cells were with integrin were used at a of and was at are as number and number in a of K562 cells and LFA-1 cells were with integrin were used at a of and was at are as K562 cells not β2 integrin gene products and are to the of E. coliα-hemolysin or A. actinomycetemcomitans leukotoxin (12Simpson D.L. Berthold P. Taichman N.S. Infect. Immun. 1988; 56: 1162-1166Crossref PubMed Google Scholar). a K562 cell showed no of to either toxin However, when K562 cells were with CD11a and CD18 genes on the same the cell line was sensitive to both RTX toxins The of the RTX toxins be by of cells with monoclonal antibody not by an isotype antibody. these K562 cells incubated with A. in and with and are to cells cells showed after toxin of the cells an in with and The of cells showed no of RTX cells with (10 were to controls not We have used and gene experiments to that A. and E. coli bind to integrin on human target cells. LFA-1 is on and not on cells of A member of the β2 integrin LFA-1, mediates binding to cells with several members of the family Springer T.A. Immunol. 1990; PubMed Scopus Google Scholar), a to during LFA-1 is in cell such as adhesion of cells to their target cells, cell and cells and cells. The host cell membrane that an for and several members of the integrin family have to as for a of Van Trends Microbiol. 1994; Full Text PDF PubMed Scopus Google Scholar). a β2 integrin that binds the is used by several that bind to are into the immune cell by the integrin and such as or with by the R.A. J. Immunol. 1990; Google Scholar). β2 LFA-1 not EMBO J. 1992; PubMed Scopus Google Scholar). The study a RTX toxins in a β2 integrin is host immune cells binding of A. actinomycetemcomitans leukotoxin to the LFA-1 the studies in this integrin as a receptor for RTX toxins. bound both CD11a and CD18 subunits when by the several into the The antibody a at 100 and a at kDa. The presence of the two β2 integrin described previously F. P. S. Springer T.A. J. PubMed Scopus Google Scholar, C. C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and the polypeptide and precursor of the that the leukotoxin is to bind to LFA-1 containing the precursor of of the with antibody showed of present in the from the toxin when with of the experiments are to this binding of CD18 to the of CD11a from the toxin or of CD11a during or such that antibody is The of an integrin receptor as the receptor for RTX toxins of the that molecular may be in the cell A of is their to that to the for such is not a of can 1992; Full Text PDF PubMed Scopus Google Scholar) of the of the integrin (16Robinson M.K. Andrew D. Rosen H. Brown D. Ortlepp S. Stephens P.X. Butcher E.C. J. Immunol. 1992; 148: 1080-1085PubMed Google Scholar). The with LFA-1 to be and at least Y. Immunol. 1990; Full Text PDF PubMed Scopus Google Scholar). is that N. D. D.R. J. Cell Biol. 1991; PubMed Scopus Google Scholar, M. F. M. J. Immunol. 1988; Google Scholar) to RTX toxins. The studies that the of the integrin may the LFA-1 on cells is in a S. Stephens P.E. Hogg N. M.K. Eur. J. Immunol. 1995; PubMed Scopus Google Scholar). cells are sensitive HL60 to both RTX toxins when with HL60 cells the same the of are the that the cells not bind either or antibodies that have to We are the in toxin are to an integrin binding in cells or to to LFA-1 of the that integrin may have on cell that RTX toxins not bind in a manner to the 3 inhibited the target cell of both RTX toxins. studies S. Stephens P.E. Hogg N. M.K. Eur. J. Immunol. 1995; PubMed Scopus Google Scholar) have that binding of to cells LFA-1 by receptor binding to RTX toxins bind to LFA-1 at the binding cells to have to both toxins. of to inhibit RTX indicates that RTX toxins of LFA-1 not in Kieba and E. T. RTX toxins are into two their target cell A. actinomycetemcomitans leukotoxin kills a narrow range of target cell cells and from man and N.S. J. Res. PubMed Scopus Google Scholar, N.S. D.L. S. M. J. J. Microbiol. Immunol. PubMed Scopus Google Scholar). The of with the range of cells actinomycetemcomitans E. coli is an RTX that is to a range of cell such as cells in and mouse S.J. Microbiol. PubMed Google Scholar). the present the that and CD18 monoclonal antibodies inhibited both toxins suggests that of LFA-1 was for However, the of E. coli to kill a range of cells may in ability to bind cell surface We are this The that cell surface receptor in the of bacterial toxins in to the RTX toxin receptor (1Pattus F. Massotte D. Wilmsen H.U. Lakey J. Tsernoglou D. Tucker A.X. Parker M.W. Experientia. 1990; 46: 180-192PubMed Google Scholar, Rie J. Jansens S. Hofte H. Degheele D. Van Mellaert H. Appl. Environ. Microbiol. 1990; 56: 1378-1385Crossref PubMed Google Scholar, Rie J. Jansens S. Hofte H. Degheele D. Van Mellaert H. Eur. J. Biochem. 1989; 186: 239-247Crossref PubMed Scopus (280) Google Scholar, E.A. Hol W.G. Curr. Opin. Struct. Biol. 1995; 5: 165-171Crossref PubMed Scopus (245) Google Scholar). of cell surface by RTX toxins the of a process in the toxin from an to an protein toxins be in a to the of biological this toxins are into and these are in such a that the of the toxin ultimately with the cell membrane is when the protein is in J.M. Lakey Pattus F. 1991; PubMed Scopus Google Scholar). is a that toxin binding to LFA-1 a to this and a of the molecule leading to of in a that a member of the LFA-1, is a cell surface receptor for RTX toxins on human target cells. The ability of β2 integrin monoclonal antibodies to inhibit cell by RTX toxins and of β2 integrin gene to a cell to a toxin-sensitive for β2 to RTX-mediated cytotoxicity. of by either A. actinomycetemcomitans or E. coli of the adhesion The ability of these to these cell adhesion to host immune cells at to study of the mechanisms in RTX toxin will a of the of this of toxins and to and was at the University of and Cell in by the and a We and N. for with the studies and D. of University of for and in of the