INFECTION AND IMMUNITY, June 1990, p. 1774-1781
Vol. 58, No. 6
0019-9567/90/061774-08$02.00/0 Copyright C) 1990, American Society for Microbiology
Demonstration of Cross-Reactivity between Bacterial Antigens and Class I Human Leukocyte Antigens by Using Monoclonal Antibodies to Shigella flexneri KRISTINA M. WILLIAMS* AND RICHARD B. RAYBOURNE
Division of Microbiology, Food and Drug Administration, Washington, D.C. 20204 Received 8 January 1990/Accepted 12 March 1990
Bacterial envelope proteins which share immunodeterminants with the human leukocyte antigen (HLA) class I histocompatibility antigen HLA-B27 may invoke spondyloarthritic disease through the process of molecular mimicry in patients expressing this phenotype. Monoclonal antibodies generated by the immunization of BALB/c mice with envelope proteins of Shigella flexneri type 2a were tested for reactivity against cultured lymphoblastoid cell lines of defined HLA phenotype. As measured by flow microfluorometry, four immunoglobulin M monoclonal antibodies reacted preferentially with HLA-B27-positive lymphocytes (HOM-2, MM) as compared with a B27-loss mutant line (1065) or cells lacking major histocompatibility complex class I antigen (Daudi, K562). Monoclonal antibodies also reacted with mouse EL-4 cells transfected with and expressing the HLA-B7 gene. Western immunoblot analysis of isolated enterobacterial envelopes demonstrated that the reactive epitope was present on bacterial proteins with an apparent relative molecular mass of 36 and 19 kilodaltons. The structural basis for the cross-reactivity of bacterial antigen and HLA-B27 appeared to reside in the portion of the HLA molecule that is responsible for allotypic specificity (amino acids 63 through 83), since monoclonal antibodies were positive by enzyme-linked immunosorbent assay with synthetic polypeptides corresponding to this segment.
Most patients who experience arthritic sequelae after enterobacterial infection express the human leukocyte antigen (HLA) class I histocompatibility antigen HLA-B27 (7). Reactive arthritis and Reiter's syndrome are often preceded by gastroenteritis involving specific gram-negative pathogens, most notably Shigella, Salmonella, Yersinia, and Campylobacter species (8). Although an antecedent episode of infection is not characteristic of ankylosing spondylitis, chronic habitation of the bowel, particularly by Klebsiella pneumoniae, is suspected (21). Of the theories proposed to explain the expression of a particular MHC haplotype and susceptibility to postinfectious spondyloarthritic lesions, molecular mimicry emerges as a potential pathogenic mechanism. Autoreactive humoral and cellular immune responses may be induced by persistent bacterial antigens sharing cross-reactive epitopes with the mimicked host protein, in this case the HLA-B27 molecule. Serologic cross-reactivity between class I antigens and arthritogenic bacteria has been demonstrated with polyvalent rabbit and human antisera and monoclonal anti-HLAB27 antibodies. How'ever, a review of previous studies reveals descrepant results between laboratories, even when similar reagents and techniques are used. For example, using sera from rabbits immunized with lymphocytes from HLAB27-positive subjects, Welsh et al. (42) demonstrated immunologic cross-reactivity against several gram-negative enteric pathogens, including K. pneumoniae and Yersinia enterocolitica; however, Archer (3), using the same antisera, failed to confirm these results. To overcome problems associated with high levels of nonspecific binding inherent to the use of whole antisera, several groups, including our own, have used two well-characterized monoclonal anti-HLA-B27 antibodies (B27.M1 and B27.M2) to identify cross-reactive bacterial epitopes. Van Bohemen et al. (38) identified a *
16-kilodalton (kDa) envelope component of K. pneumoniae and Y. enterocolitica which reacted with B27.M1 and a B27.M2-reactive 20-kDa antigen of Shigella flexneri 2a. Ogasawara et al. (25) also used B27.M2 to demonstrate cross-reactivity with 80- and 60-kDa antigens of K. pneumoniae K43. No cross-reactivity was seen with seven other enterobacterial isolates, including S. flexneri, Y. enterocolitica, or the K77 strain of K. pneumoniae. In prior studies (30), we found B27.M2- and B27.M1-cross-reactive antigens of 36 and 23 kDa that were common to many gram-negative bacteria, not all of which have been etiologically associated with reactive arthritis. The present study was undertaken because of the potential significance of molecular mimicry in the initiation of characteristic spondyloarthritic lesions and because of the previous problems associated with identification of the relevant bacterial antigen. Rather than using HLA-B27 monoclonal or polyclonal antisera to identify these antigens, we produced monoclonal antibodies against an isolate of S. flexneri 2a that was cultured from a patient with Reiter's syndrome. These antibodies were then used to demonstrate the existence of cross-reactive epitopes on the native HLA molecule and on synthetic polypeptides corresponding to the HLA-B variable region. MATERIALS AND METHODS
Bacterial strains. Bacterial isolates were generously provided as follows: S. flexneri, Shigella sonnei, and Salmonella sp. from J. G. Wells, Centers for Disease Control, Atlanta, Ga.; Escherichia coli, K. pneumoniae, and Yersinia pseudotuberculosis from D. T. Y. Yu, University of California, Los Angeles, School of Medicine; and Y. enterocolitica from Peter Sheldon, Public Health Laboratory Service Board, London, England. Bacterial envelope preparations. Whole bacterial envelopes were prepared by ultrasonication and differential cen-
Corresponding author. 1774
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CROSS-REACTIVITY OF BACTERIAL ANTIGEN AND HLA
TABLE 1. HLA phenotype of cell lines used in testing monoclonal antibodies
HOM-2 Daudi K562 MM 1065 EL-4 (mouse)
EB-lb EA-6b a
b
TABLE 2. Specificity of monoclonal antibodies for cultured cell lines Specificity for the indicated celi line
MHC class I surface antigena
Cell line
1775
Cell line HLA-A
HLA-B
HLA-C
3
27
1
2,24 24
27,44
2,5 2
7 2
MHC, Major histocompatibility complex. Transfected mouse cell line (14).
trifugation as described previously (29). Total protein content was determined by the method of Lowry et al., modified for membrane-associated proteins (24). Production of monoclonal antibodies. BALB/c female mice were inoculated in multiple subcutaneous sites with 100 ,ug of total protein suspended in 0.1 ml of normal saline. Mice were rested for 1 month and then given a final intraperitoneal inoculum of 100 ,ug of total protein. The immunogen for all fusions was whole bacterial envelopes prepared from S. flexneri 2a isolated from a patient with Reiter's syndrome (SSU 6335). Splenocytes were fused with syngeneic plasmacytoma cells P3x63.Ag8.653) as described by Kohler and Milstein (19) and modified by Kennett (17) and Oi and Herzenberg (26), with polyethylene glycol 1500 (BoehringerMannheim Biochemicals, Indianapolis, Ind.) as the fusogen. Positive hybridomas were cloned by using the Epics CS flow cytometer equipped with an autoclone accessory. Antibodies were isotyped by radial immunodiffusion by using rabbit anti-mouse subclass-specific antisera (Binding Site, Inc., San Diego, Calif.). Ascites fluids were produced as previously described (15). Immunoglobulin M (IgM) was purified from ascites fluids by Sephadex G200 (Pharmacia Fine Chemicals, Piscataway, N.J.) chromatography followed by hypoosmotic precipitation by dialysis of IgM-containing fractions against 1 mM Tris hydrochloride (pH 7.5). Synthetic polypeptides. Peptides were synthesized in an automated peptide synthesizer (model 430A; Applied Biosystems, Inc., Foster City, Calif.). Peptide from the solidphase support was deprotected and released by treating the protected resin-attached peptide with anhydrous hydrofluoric acid containing 10% anisole for 2 h at 0°C. Peptide purity was monitored by reversed phase high-pressure liquid chromatography. Peptides were determined to be >90% pure. ELISA. Hybridomas were screened for antibody production by an enzyme-linked immunosorbent assay (EUSA) with 0.05 p.g total bacterial envelope protein applied to each well of 96-well flat-bottomed polystrene microdilution plates (Immulon I; Dynatech Laboratories, Inc., Chantilly, Va.). Hybridoma supernatants (100 1d) were pipetted into coated wells and incubated for 2 h at room temperature. Bound antibody was detected with a 1:1,000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (heavy and light) (Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.) with p-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, Mo.) as the chromogen. ELISAs with synthetic polypeptide were optimized to 10 ,ug/well of peptide bound to Dynatech Immulon II microdilution plates. The ELISAs were carried out as described above by using 100 1.l of a 2-,ug/ml solution of purified IgM suspended in 1% normal goat serum.
A7E2
HOM-2 Daudi K562 MM 1065 EL-4 EB-1 EA-6
C7E1O
E2B6
FlOE10
D1D8a
-
+
+
+
-
-
-
-
-
+
+
+
+
-
+ -
+ _
+ _
+ -
+
+
+
+
_ _
-
-
-
-
-
+
a Isotype control.
Flow cytometry. Reactivity of monoclonal antibodies toward native HLA molecules was assessed by indirect immunofluorescence with transformed human lymphoblastoid cell lines or transfected murine cells that express HLA surface antigen. Daudi and K562 cell lines were obtained from American Type Culture Collection, Rockville, Md. HOM-2 and MM cells (HLA-B27 positive) and an MMderived HLA-B27 negative mutant (1065) were provided by D. T. Y. Yu. The murine T-cell iymphoma line (EL-4) and EL-4-derived transfectants (EA-6, EB-1) were obtained from Victor H. Engelhard, University of Virginia School of Medicine, Charlottesville, Va. (14). All cells were maintained in RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and nonessential amino acids. Transfected cells were maintained in the same medium that also contained 250 ,ug of G418 (GIBCO Laboratories, Grand Island, N.Y.) per ml. Methods for fluorescent staining of live cells were performed as previously described (30) with a 1:5 to 1:10 dilution of hybridoma supernatant and a 1:10 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgM F(ab')2 fragments (Jackson). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis. Cultured lymphocytes (108) were washed three times by centrifugation for 5 min at 500 x g in 0.4 M NaCl-200 mM Tris hydrochloride (pH 7.4). Membrane proteins were extracted by resuspending the cell pellet in 1 ml of the same buffer containing 0.5% Nonidet P-40 (Pierce Chemical Co., Rockford, Ill.) and 1 mM phenylmethylsulfonyl fluoride (Sigma). After incubation for 30 min on ice, extracted cells were pelleted for 1 h at 40,000 x g. Supernatant samples containing 21 ,ul of membrane extract (40 to 50 pg of total protein) were examined by electrophoresis under reducing conditions by the method of Laemmli (22) with a 12.5% polyacrylamide resolving gel. Alternatively, pelleted bacterial envelopes were suspended in sample buffer, and 25-,ul samples (50 ,ug of total protein) were electrophoretically separated under the same conditions. Methods for transfer of proteins to nitrocellulose membranes were as described earlier (34). After blocking for 1 h with 1% bovine serum albumin (radioimmunoassay grade; Sigma) in 0.04 M Tris-0.5 M NaCl (pH 7.5), reactive bands were observed by using a 1:5 to 1:10 dilution of hybridoma supernatant and a 1:1,500 dilution of peroxidaseconjugated goat anti-mouse IgM. Blots were developed with a solution of 0.05% 4-chlor-1-naphthol ard 0.006% H202. RESULTS Antibody-secreting hybridomas were identified by ELISA with envelope proteins of S. flexneri 2a as the solid-phase
INFECT. IMMUN.
WILLIAMS AND RAYBOURNE
1776
U)
LUI LI.
0
LU
z
iq
Lu
I.
;j,i~
#
1-I LU
if. D1D8
LOGIo FLUORESCENCE INTENSITY FIG. 1. Immunofluorescent analysis of cultured lymphocytes and transfected murine cells with monoclonal antibodies. Cells were stained by an indirect method using hybridoma supernatants followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgM F(ab')2 fragments as described in Materials and Methods. HLA-cross-reactive antibodies (A7E2, FlOE10) are compared with the non-cross-reactive isotype control (D1D8).
reactant. Culture fluids with an A405 of >1.0 were then evaluated for specificity of binding to the cultured lympho-
cytes listed in Table 1. Of more than 600 antibacterial antibodies tested by flow microfluorometry, 4 were selected for further characterization, based on intensity of surface fluorescence observed when live cells were stained by an
indirect method. These four antibodies (designated A7E2, C7E10, E2B6, and FlOE10) were all of the IgM isotype. An additional antibacterial IgM monoclonal antibody, designated D1D8, was randomly chosen for use as an isotype control. Results of flow cytometric assays are summarized in Table
VOL. 58, 1990
CROSS-REACTIVITY OF BACTERIAL ANTIGEN AND HLA
1777
TABLE 3. Sequences of MHC class I-specific synthetic polypeptides (variable region) Amino acid sequence
Peptide 65
HLA-B*2705a HLA-B7 HLA-B40
70
E
T
N
T
Q Q
I
E
T
Q
I
I
C Y S
75
80
K
A
K
A
D
R
E
D
L
R
T
L
L
A
Q
A
Q Q
T
K
T
D
R
E
L
R
N
L
R
G
K
A
N
T
Q
T
Y
R
E
S S
L
R
N
L
R
G
R
a Formerly designated HLA-B27.1. Contains sequence required for B27.M2 binding (5).
2. HOM-2 cells, which are homozygous at the HLA-B locus, were strongly positive when stained with a saturating concentration of each of the four test monoclonal antibodies. By contrast, Daudi and K562 cells, which lack cell surface expression of class I molecules, were uniformly negative (Fig. 1). MM cells displayed a level of surface fluorescence comparable to that of HOM-2 cells. The mutant cell line 1065, which was derived from the HLA-B27-positive cell line MM but does not express for HLA-B locus antigens, was also tested. Although the antibodies stain MM cells more brightly, the 1065 cells demonstrated observable levels of fluorescence staining (Fig. 1). The transfected cell line EB-1, which expresses surface HLA-B7, was also positive with all four test monoclonal antibodies; however, cells transfected with and expressing HLA-A2 (EA-6) and the parental cell line (EL-4) were negative (Fig. 1). To characterize the molecular basis for antibody activity, synthetic polypeptides were produced with an amino acid sequence identical to the variable region-spanning residues 63 through 83 of the HLA-B*2705, HLA-B7, and HLA-B40 heavy chains (Table 3). Monoclonal antibodies were tested for antipeptide activity by ELISA. All antibodies were positive with each of the synthetic polypeptides, although preferential binding was observed with the HLA-B*2705 peptide and the homologous antigen (S. flexneri 6335 enve-
lope) (Fig. 2). Antibodies were nonreactive with two synthetic peptides of unrelated sequence, which were included as negative controls (data not shown). Membrane proteins of the HLA-B27-positive cell line MM were extracted with detergent, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose for Western blot analysis with the HLAreactive monoclonal antibody (Fig. 3). All monoclonal antibodies bound to a protein with a molecular mass of approximately 44 kDa, which is identical to that of the MHC class I heavy chain. A protein of similar molecular mass was identified by the B27-specific IgM monoclonal antibody B27.M2, which was included as a positive control. Other isotype controls were nonreactive by Western blotting. When tested by immunoblotting with electrophoretically resolved S. flexneri 2a membrane proteins, each of the four HLA-reactive monoclonal antibodies gave an identical pattern of reactivity. A major reactive protein was observed at 36 kDa, and a less prominent band was seen at 19 kDa. A I 1
B
C
D
E
F
G
6
84 58
SPECIFICITY OF ANTI-BACTERIAL ANTIBODIES
48.5
(Antibody Concentration * 2 ug/ml)
O.D. 406 nm
36.5
1.5 26.6
0.5
A7E2
W
C7E1O
HLA-B27 peptide HLA-B40 peptide
E2B6
FlOE10
D1D8
HLA-B7 peptide S. flexneri 6335
FIG. 2. Reactivity by ELISA of antibacterial monoclonal antibodies with HLA-B synthetic polypeptides and S. flexneri envelope proteins. Peptides were synthesized according to the sequence of their respective variable regions (amino acids 63 through 83). Assays (described in Materials and Methods) used monoclonal antibodies purified from ascites fluids. Cell-reactive monoclonal antibodies were positive against all three synthetic polypeptides, although stronger reactivity was observed with HLA-B27 peptide and bacterial envelope proteins. Control antibacterial monoclonal antibodies (D1D8 isotype control) were negative against synthetic peptides.
FIG. 3. Western blot analysis of detergent-extracted MM cells (HLA-B27 positive) with monoclonal antibodies. Cell membrane proteins were extracted with Nonidet P-40 and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis as described in Materials and Methods. Antibacterial monoclonal antibodies (lanes B through E) and the HLA-B27-specific monoclonal antibodies, B27.M2 (lane F), reacted with a protein with a molecular mass consistent with that of the major histocompatibility complex class I H chain (44 kDa). Lanes: A, Coomassie blue-stained membrane extract; B, A7E2; C, C7E10; D, E2B6; E, FlOE10; F, B27.M2; G, D1D8 (isotype control).
1778
INFECT. IMMUN.
WILLIAMS AND RAYBOURNE A
3
C
D
E
F
84
588
.....
48.5
*_9MOW-' 4f,W
AII
26.6
FIG. 4. Western blot analysis of S. flexneri envelope proteins with monoclonal antibodies. All of the HLA-reactive monoclonal antibodies reacted with proteins of 36 and 19 kDa; the 36-kDa protein was the most prominent. Control antibacterial monoclonal antibodies gave a different pattern of reactivity. Lanes: A, Coomassie blue-stained envelope proteins; B, A7E2; C, C7E10; D, E2B6; E, FlOE10; F, D1D8 (isotype control).
Neither protein reacted with antibody D1D8, which was included as an isotype control (Fig. 4). To determine whether the cross-reactive epitope was unique to this isolate of S. flexneri, envelope proteins were prepared from a collection of S. flexneri strains representing most serotypes and from a group of related bacteria, some of which are also epidemiologically associated with arthritic disease. These preparations were standardized for protein concentration and tested by ELISA with the monoclonal antibody. Positive activity against bacterial strains was observed with the HLA-reactive monoclonal antibodies (Table 4). The isotype control was specific for S. flexneri 2a. Although the reaction of antibody C7E10 was weaker than that of the remaining three antibodies, no preferential binding was seen with any strains tested.
DISCUSSION Despite the epidemiologic evidence associating gram-negative members of the family Enterobacteriaceae with the seronegative spondyloarthropathies, the mechanism by which bacterial factors can initiate and perpetuate these diseases remains hypothetical. Furthermore, in contemplating pathogenic mechanisms, equal consideration must be given to genetic factors, such as the presence of the class I histocompatibility antigen HLA-B27, which undoubtedly predisposes individuals to postinfectious arthritic sequelae (6). The results presented in this report demonstrate that monoclonal antibodies prepared against an arthritogenic organism also recognize epitopes present on class I histocompatibility antigen. This supports the hypothesis that the link between environmental and genetic etiologic factors is a common immunodeterminant and that this molecular mimicry could be of pathologic significance in the induction of chronic arthritic disease. The four cross-reactive monoclonal antibodies were initially identified by using cultured cell lines in an indirect microfluorometric assay. Although previous investigators have used a microcytotoxicity assay to determine HLA
specificity of their monoclonal antibodies (20), we tried to avoid any potential problems associated with complementbinding capabilities of the monoclonal antibodies. The four monoclonal antibodies had similar patterns of reactivity against the cultured cell lines. Antibodies were completely negative against cells lacking MHC class I antigens (Daudi, K562, EL-4) and against murine lymphoid cells transfected with and expressing only HLA-A2 (EA-6). Monoclonal antibodies were not, however, singularly positive with HLAB27-positive cells; they were equally reactive with transfectants expressing only HLA-B7 (EB-1) and nominally reactive with cells lacking HLA-B antigen but expressing HLA-A24 and HLA-C2 (1065). This low degree of allelic specificity indicates that the epitope recognized by these antibodies consists of nonpolymorphic residues shared by several HLA class I antigens. We can also conclude that this epitope differs from that recognized by B27.M2, which is specific for B27 and Bw47 (5). Although it appears that our monoclonal antibodies bind preferentially with B-locus antigens, fluorescence intensity between cultured lymphocytes may not be directly comparable, because the surface density of HLA antigen may vary from one cell line to another. In Western blot analyses against detergent-extracted MM (HLA-B27-positive) cells, all four cross-reactive monoclonal antibodies bound to a single band at an approximate molecular mass of 44 kDa. This molecular mass corresponds to that previously reported for the MHC class I heavy chain and is identical to that observed when monoclonal antibody B27.M2 is subjected to immunoblot analysis. Based on ELISA results with synthetic polypeptides corresponding to amino acids 63 through 83 of the class I heavy chain, we determined that the epitope recognized by our monoclonal antibodies was present in this segment. Previous investigators have shown that this polymorphic sequence also contains the epitope responsible for specific binding of B27.M2 (5) and recognition structures for allospecific cytotoxic T lymphocytes (2, 41). Although this segment would appear critical in defining allospecificity of the HLA-B27 molecule, significant structural homology exists between the HLA-B27 variable region and the corresponding segment of nonHLA-B27 class I molecules (32). Several nonpolymorphic epitopes within the variable region have been identified by using monoclonal antibodies generated against lymphocytic cells (1, 11, 27, 28, 31). The existence of such epitopes would explain the reactivity of our four monoclonal antibodies with non-HLA-B27 class I antigen and in particular with the HLA-B7 and HLA-B40 synthetic polypeptides, which were also derived from their respective variable regions. The presence of such epitopes may also explain the high incidence of HLA-B7 CREG (cross-reactive group) antigens in HLA-B27-negative spondyloarthritic patients (4, 18). Using S. flexneri envelope proteins, we observed qualitatively identical patterns of immunoblot reactivity for the four monoclonal antibodies. The reactive epitope was present on 36- and 19-kDa antigen of S. flexneri, but was not restricted to this species of the family Enterobacteriaceae. Strong ELISA activity was observed with all strains tested, even with species generally considered nonarthritogenic (e.g., E. coli). Despite the presence of such antigens, additional bacterial virulence mechanisms (such as the ability to invade host cells and survive intracellularly) are also characteristic of species with arthritogenic potential. Indeed, the ability of an organism to establish infection is a prerequisite for host exposure to and immune system recognition of cross-reactive antigens. The occurrence of a cross-reactive epitope that is present on many organisms is consistent with the
VOL. 58, 1990
CROSS-REACTIVITY OF BACTERIAL ANTIGEN AND HLA
1779
TABLE 4. Reactivity of monoclonal antibodies with enterobacterial envelope proteins
A405
Bacterial strain A7E2
C7E1O
E2B6
FlOE10
D1D8I
la 1235-66 la 1921-71 lb 6115b lb 4343-70 lb 5612-73 2a 6335b 2a 947b 2a 2747-71 2a 29903 2b 12022 2b 1676 2b 9768 3 2146-66 3 2783-71 4a 6603-63 4a 1862-71 4b 880-69 4b 1242-70 5 1170-74 5 5103-82 6 2924-71 6 796-83
1.845 1.484 1.801 1.223 1.328 1.909 1.795 1.505 0.920 1.639 1.437 1.362 1.430 1.557 1.570 1.760 1.337 1.002 1.164 1.166 1.163 1.132
0.652 0.597 0.411 0.454 0.478 0.517 0.406 0.574 0.269 0.470 0.430 0.297 0.486 0.568 0.531 0.454 0.445 0.347 0.406 0.441 0.428 0.429
1.888 1.604 1.115 1.172 1.343 1.527 1.679 1.508 1.070 1.316 1.410 1.023 1.564 1.586 1.484 1.517 1.426 1.036 1.038 1.149 0.996 1.030
1.285 1.384 1.265 1.012 1.439 1.420 1.397 1.167 0.653 1.496 1.024 1.011 1.115 1.163 1.193 1.296 1.907 1.276 1.804 1.496 1.332 1.532
0.086 0.057 0.056 0.050 0.048 1.836 1.947 1.735 1.389 0.075 0.054 0.049 0.051 0.052 0.049 0.070 0.072 0.058 0.048 0.063 0.055 0.059
Shigella sonnei 6425 7063
1.754 1.890
0.449 0.602
1.933 1.740
1.226 1.341
0.071 0.055
Salmonella heidelburg 7375b
1.204
0.434
1.342
1.204
0.085
Salmonella typhimurium 24b
1.690
0.511
1.872
1.226
0.078
Klebsiella pneumoniae K43
1.289
0.337
1.365
0.760
0.058
Escherichia coli 10908 3374
1.835 1.995
0.611 0.655
1.745 1.885
1.329 1.526
0.064 0.054
1.993 1.180
0.698 0.425
1.992 1.174
1.686 0.946
0.066 0.951
1.289 1.567
0.577 0.763
1.496 1.926
0.855 1.948
0.054 0.074
Shigella flexneri
Yersinia pseudotuberculosis 78 b
269b
Yersinia enterocolitica
A2b A6b
a Isotype control. b Isolated from a patient with Reiter's syndrome or reactive arthritis.
finding that many species of gram-negative bacteria are epidemiologically implicated in these diseases. Furthermore, many of the 36- to 38-kDa major outer membrane proteins of members of the family Enterobacteriaceae are conserved phylogenetically and considered common antigens (13), making them candidate recognition structures for our crossreactive monoclonal antibodies. A comparison of the relative mobility in polyacrylamide gels of the bacterial antigen recognized by B27.M2 indicates that our monoclonal antibodies have a different antigen specificity. The molecular specificity of our antibodies is currently under investigation. Further studies on humoral and cell-mediated responses of spondyloarthritic patients which parallel the course of their disease are required to determine the pathologic significance of the 36- and 19-kDa antigens. A vigorous antibody response against the causative organism is present in all patients after a gram-negative enteritis; however, those who
develop subsequent arthritis show higher and more persistent antibody titers in serum (35, 36, 39). Serum antibodies of the IgG, IgM, and IgA classes directed against Yersinia proteins of 35 to 36 kDa have been reported in patients with reactive arthritis after infection with Y. enterocolitica (12, 33, 40), but whether this antigen corresponds to the 36-kDa antigen recognized by our cross-reactive monoclonal antibody is yet to be determined. The availability of monoclonal antibodies will facilitate purification of this antigen for further studies with patient specimens. Of greater interest is the potential regulatory role of bacterially induced HLA class I-reactive antibodies on Tcell responses in a susceptible host. Several monoclonal anti-class I HLA antibodies, in the presence of adherent antigen-presenting cells, can inhibit mitogen- and antigeninduced T-cell proliferation (9, 10, 37). An antibody-mediated down-regulation of cellular responses to arthritogenic
1780
WILLIAMS AND RAYBOURNE
bacteria could facilitate persistence of bacteria in infected cells. Indeed, defective cellular immune functions have been reported in arthritic patients. Leino et al. (23) found that lymphoproliferative response to bacterial antigen was depressed in the peripheral blood mononuclear cells of patients who developed post-Yersinia reactive arthritis as compared with those who recovered from yersiniosis uneventfully. The impaired response was not organism specific, since significant inhibition was obtained after stimulation with either Yersinia or E. coli antigen. Similarly, peripheral blood mononuclear cells from patients with reactive arthritis after Salmonella typhimurium infection showed an impaired lymphoproliferative response to in vitro stimulation with the causative organism (16). Hypothetically, HLA-B27-reactive antibodies could be induced by a common bacterial antigen in all infected patients, but their negative regulatory effects would be expressed only in patients who are HLA-B27 positive or whose class I antigens contain the cross-reactive
epitope. In conclusion, we identified 36- and 19-kDa bacterial envelope proteins that share cross-reactive epitope(s) with class I histocompatibility molecules. Although the mechanism by which these or other bacterial antigens precipitate joint disease remains unknown, the existence of shared immunodeterminants lends support to the concept of molecular mimicry in disease induction.
LITERATURE CITED 1. Antonelli, P., B. Nisperos, P. Gladstone, and J. Hansen. 1983. Recognition of a unique HLA-B27.7, w22, 17, 14, w63, w46 common epitope by two independently derived BALB/c monoclonal antibodies. Hum. Immunol. 8:296. 2. Aparicio, P., M. A. Vega, and J. A. Lopez de Castro. 1985. One allogeneic cytolytic T lymphocyte clone distinguishes three different HLA-B27 subtypes: identification of amino acid residues influencing the specificity and avidity of recognition. J. Immunol. 135:3074-3081. 3. Archer, J. R. 1981. Search for cross-reactivity between HLAB27 and Klebsiella pneumoniae. Ann. Rheum. Dis. 40:400-403. 4. Arnett, F. C., Jr., M. C. Hochberg, and W. B. Bias. 1977. Cross-reactive HLA antigens in B27 Reiter's syndrome and sacroiliitis. Johns Hopkins Med. J. 141:193-197. 5. Bjorkman, P. J., M. A. Saper, B. Samraoui, W. S. Bennett, J. L. Strominger, and D. C. Wiley. 1987. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature (London) 329:512-518. 6. Brewerton, D. A., M. Caffrey, F. D. Hart, D. C. 0. James, A. Nichols, and R. D. Sturrock. 1973. Ankylosing spondylitis and HLA-27. Lancet i:904-907. 7. Brewerton, D. A., M. Caffrey, A. Nicholls, D. Walters, J. K. Oates, and D. C. James. 1973. Reiter's disease and HLA-27. Lancet i:996-998. 8. Bunning, V. K., R. B. Raybourne, and D. L. Archer. 1988. Foodborne enterobacterial pathogens and rheumatoid disease. J. Appl. Bacteriol. Symp. Suppl. 65:87S-107S. 9. Dasgupta, J. D., K. Cemach, D. P. Dubey, E. J. Yunis, and D. B. Amos. 1987. The role of class I histocompatibility antigens in the regulation of T-cell activation. Proc. Natl. Acad. Sci. USA
84:1094-1098.
10. Dasgupta, J. D., and E. J. Yunis. 1987. Receptor-like role of HLA-class I antigens: regulation of T cell activation. J. Immunol. 139:672-677. 11. Ellis, S., C. Taylor, and A. McMichael. 1982. Recognition of HLA-B27 and related antigens by a monoclonal antibody. Hum. Immunol. 5:49-59. 12. Gronberg, A., A. Fryden, and E. Kihlstrom. 1989. Humoral immune response to individual Yersinia enterocolitica antigens in patients with and without reactive arthritis. Clin. Exp. Immunol. 76:361-365. 13. Hofstra, H., and J. Dankert. 1980. Major outer membrane
INFECT. IMMUN.
proteins: common antigens in Enterobacteriaceae species. J. Gen. Microbiol. 119:123-131. 14. Holterman, M. J., and V. H. Engelhard. 1986. HLA antigens expressed on murine cells are preferentially recognized by murine cytotoxic T cells in the context of the H-2 major histocompatibility complex. Proc. Natl. Acad. Sci. USA 83: 9699-9703. 15. Hoogenraad, N. J., and C. J. Wraight. 1986. The effect of pristane on ascites tumor formation and monoclonal antibody production. Methods Enzymol. 121:375-381. 16. Inman, R. D., B. Chiu, M. E. A. Johnston, S. Vas, and J. Falk. 1989. HLA class I-related impairment in IL-2 production and lymphocyte response to microbial antigens in reactive arthritis. J. Immunol. 142:4256-4260. 17. Kennett, R. H. 1979. Cell fusion. Methods Enzymol. 58:345359. 18. Khan, M. A. 1983. B7-CREG and ankylosing spondylitis. Br. J. Rheumatol. 22(Suppl. 2):129-133. 19. Kohler, G., and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256:495-497. 20. Kono, D. H., M. Ogasawara, R. B. Effros, M. S. Park, R. L. Waldord, and D. T. Y. Yu. 1985. Ye-1, a monoclonal antibody that cross-reacts with HLA-B27 lymphoblastoid cell lines and an arthritis causing bacteria. Clin. Exp. Immunol. 61:503-508. 21. Kuverski, T. T., M. G. Morse, R. G. Rate, and M. D. Bonnell. 1983. Increased recovery of Klebsiella from the gastrointestinal tract of Reiter's syndrome and ankylosing spondylitis patients. Br. J. Rheumatol. 22(Suppl. 2):85-90. 22. Laemmli, U. K. 1979. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 23. Leino, R., R. Vuento, S. Koskimies, M. Viander, and A. Toivanen. 1983. Depressed lymphocyte transformation by yersinia and Escherichia coli in yersinia arthritis. Ann. Rheum. Dis. 42:176-181. 24. Markwell, M. A., S. M. Haas, L. L. Bieber, and N. E. Tolbert. 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87:206-210. 25. Ogasawara, M., D. H. Kono, and D. T. Y. Yu. 1986. Mimicry of human histocompatibility HLA-B27 antigens by Klebsiella pneumoniae. Infect. Immun. 51:901-908. 26. Oi, V. T., and L. A. Herzenberg. 1980. Immunoglobulin producing hybrid cell lines, p. 351-372. In B. B. Mishell and S. M. Shiigi (ed.), Selected methods in cellular immunology. 27. Parham, P., P. Antonelli, L. A. Herzenberg, T. J. Kipps, A. Fuller, and F. E. Ward. 1986. Further studies on the epitopes of HLA-B7 defined by murine monoclonal antibodies. Hum. Immunol. 15:44-67. 28. Parham, P., C. J. Barnstable, and W. F. Bodmer. 1979. Use of a monoclonal antibody (W6/32) in structural studies of HLA-A, B, C antigens. J. Immunol. 123:342-349. 29. Portnoy, D. A., H. Wolf-Watz, I. Bolin, A. B. Beeder, and S. Falkow. 1984. Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins. Infect. Immun. 43:108-114. 30. Raybourne, R. B., V. K. Bunning, and K. M. Williams. 1988. Reaction of anti-HLA-B monoclonal antibodies with envelope proteins of Shigella species: evidence for molecular mimicry in the spondyloarthropathies. J. Immunol. 140:3489-3495. 31. Rebai, N., and B. Malissen. 1983. Structural and genetic analyses of HLA class I molecules using monoclonal xenoantibodies. Tissue Antigens 22:107-117. 32. Szots, H., G. Reithmuller, E. Weiss, and T. Meo. 1986. Complete sequence of HLA-B27 cDNA identified through the characterization of structural markers unique to the HLA-A, -B, and -C allelic series. Proc. Natl. Acad. Sci. USA 83:1428-1432. 33. Toivanen, R., R. Tertti, R. Lahesmaa-Rantala, T. H. Stahlberg, and K. Granfors. 1988. Factors associated with the development of reactive arthritis. Br. J. Rheumatol. 27(Suppl. 2):46-51. 34. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose
VOL. 58, 1990
35.
36.
37.
38. 39.
CROSS-REACTIVITY OF BACTERIAL ANTIGEN AND HLA
sheets: procedures and some applications. Proc. Natl. Acad. Sci. USA 76:43504354. Truli, A. K., C. G. Eastmond, G. S. Panayi, and T. M. S. Reid. 1986. Salmonella reactive arthritis: serum and secretory antibodies in eight patients identified after a large outbreak. Br. J. Rheumatol. 25:13-19. Truli, A., A. Ebringer, G. Panayi, R. Ebringer, and D. C. 0. James. 1984. HLA-B27 and the immune response to enterobacterial antigens in ankylosing spondylitis. Clin. Exp. Immunol. 55:74-80. Turco, M. C., M. De Felice, L. Corbo, G. Morrone, R. Mertelsmann, S. Ferrone, and S. Venuta. 1985. Regulatory role of a monomorphic determinant of HLA class I antigens in T cell proliferation. J. Immunol. 135:2268-2272. Van Bohemen, C. G., F. C. Grument, and H. C. Zanen. 1984. Identification of HLA-B27M1 and -M2 cross-reactive antigens in Klebsiella, Shigella and Yersinia. Immunology 52:607-610. Van Bohemen, C. G., R. J. Lionarons, P. van Bodegom, H. J.
1781
Dinant, J. E. Landheer, A. J. J. M. Nabbe, F. C. Grumet, and H. C. Zanen. 1985. Susceptibility and HLA-B27 in post-dysenteric arthropathies. Immunology 56:377-379. 40. Van Bohemen, C. G., A. J. W. van Alphen, 0. G. Zanen-Lim, A. J. Dekker-Saeys, and H. C. Zanen. 1983. Antibodies against cell envelope antigens of Yersinia enterocolitica in reactive arthritis and ankylosing spondylitis. Br. J. Rheumatol.
22(Suppl. 2):83-84. 41. Vega, M. A., A. Ezquerra, S. Rojo, P. Aparicio, R. Bragado, and J. A. Lopez de Castro. 1985. Structural analysis of an HLA-B27 functional variant: identification of residues that contribute to the specificity of recognition by cytolytic T lymphocytes. Proc. Natl. Acad. Sci. USA 82:7394-7398. 42. Welsh, J., H. Avakian, P. Cowling, A. Ebringer, P. Wooley, G. Panayi, and R. Ebringer. 1980. Ankylosing spondylitis, HLAB27 and Klebsiella. I. Cross-reactivity studies with rabbit antisera. Br. J. Exp. Pathol. 61:85-91.
Vol. 58, No. 6
0019-9567/90/061774-08$02.00/0 Copyright C) 1990, American Society for Microbiology
Demonstration of Cross-Reactivity between Bacterial Antigens and Class I Human Leukocyte Antigens by Using Monoclonal Antibodies to Shigella flexneri KRISTINA M. WILLIAMS* AND RICHARD B. RAYBOURNE
Division of Microbiology, Food and Drug Administration, Washington, D.C. 20204 Received 8 January 1990/Accepted 12 March 1990
Bacterial envelope proteins which share immunodeterminants with the human leukocyte antigen (HLA) class I histocompatibility antigen HLA-B27 may invoke spondyloarthritic disease through the process of molecular mimicry in patients expressing this phenotype. Monoclonal antibodies generated by the immunization of BALB/c mice with envelope proteins of Shigella flexneri type 2a were tested for reactivity against cultured lymphoblastoid cell lines of defined HLA phenotype. As measured by flow microfluorometry, four immunoglobulin M monoclonal antibodies reacted preferentially with HLA-B27-positive lymphocytes (HOM-2, MM) as compared with a B27-loss mutant line (1065) or cells lacking major histocompatibility complex class I antigen (Daudi, K562). Monoclonal antibodies also reacted with mouse EL-4 cells transfected with and expressing the HLA-B7 gene. Western immunoblot analysis of isolated enterobacterial envelopes demonstrated that the reactive epitope was present on bacterial proteins with an apparent relative molecular mass of 36 and 19 kilodaltons. The structural basis for the cross-reactivity of bacterial antigen and HLA-B27 appeared to reside in the portion of the HLA molecule that is responsible for allotypic specificity (amino acids 63 through 83), since monoclonal antibodies were positive by enzyme-linked immunosorbent assay with synthetic polypeptides corresponding to this segment.
Most patients who experience arthritic sequelae after enterobacterial infection express the human leukocyte antigen (HLA) class I histocompatibility antigen HLA-B27 (7). Reactive arthritis and Reiter's syndrome are often preceded by gastroenteritis involving specific gram-negative pathogens, most notably Shigella, Salmonella, Yersinia, and Campylobacter species (8). Although an antecedent episode of infection is not characteristic of ankylosing spondylitis, chronic habitation of the bowel, particularly by Klebsiella pneumoniae, is suspected (21). Of the theories proposed to explain the expression of a particular MHC haplotype and susceptibility to postinfectious spondyloarthritic lesions, molecular mimicry emerges as a potential pathogenic mechanism. Autoreactive humoral and cellular immune responses may be induced by persistent bacterial antigens sharing cross-reactive epitopes with the mimicked host protein, in this case the HLA-B27 molecule. Serologic cross-reactivity between class I antigens and arthritogenic bacteria has been demonstrated with polyvalent rabbit and human antisera and monoclonal anti-HLAB27 antibodies. How'ever, a review of previous studies reveals descrepant results between laboratories, even when similar reagents and techniques are used. For example, using sera from rabbits immunized with lymphocytes from HLAB27-positive subjects, Welsh et al. (42) demonstrated immunologic cross-reactivity against several gram-negative enteric pathogens, including K. pneumoniae and Yersinia enterocolitica; however, Archer (3), using the same antisera, failed to confirm these results. To overcome problems associated with high levels of nonspecific binding inherent to the use of whole antisera, several groups, including our own, have used two well-characterized monoclonal anti-HLA-B27 antibodies (B27.M1 and B27.M2) to identify cross-reactive bacterial epitopes. Van Bohemen et al. (38) identified a *
16-kilodalton (kDa) envelope component of K. pneumoniae and Y. enterocolitica which reacted with B27.M1 and a B27.M2-reactive 20-kDa antigen of Shigella flexneri 2a. Ogasawara et al. (25) also used B27.M2 to demonstrate cross-reactivity with 80- and 60-kDa antigens of K. pneumoniae K43. No cross-reactivity was seen with seven other enterobacterial isolates, including S. flexneri, Y. enterocolitica, or the K77 strain of K. pneumoniae. In prior studies (30), we found B27.M2- and B27.M1-cross-reactive antigens of 36 and 23 kDa that were common to many gram-negative bacteria, not all of which have been etiologically associated with reactive arthritis. The present study was undertaken because of the potential significance of molecular mimicry in the initiation of characteristic spondyloarthritic lesions and because of the previous problems associated with identification of the relevant bacterial antigen. Rather than using HLA-B27 monoclonal or polyclonal antisera to identify these antigens, we produced monoclonal antibodies against an isolate of S. flexneri 2a that was cultured from a patient with Reiter's syndrome. These antibodies were then used to demonstrate the existence of cross-reactive epitopes on the native HLA molecule and on synthetic polypeptides corresponding to the HLA-B variable region. MATERIALS AND METHODS
Bacterial strains. Bacterial isolates were generously provided as follows: S. flexneri, Shigella sonnei, and Salmonella sp. from J. G. Wells, Centers for Disease Control, Atlanta, Ga.; Escherichia coli, K. pneumoniae, and Yersinia pseudotuberculosis from D. T. Y. Yu, University of California, Los Angeles, School of Medicine; and Y. enterocolitica from Peter Sheldon, Public Health Laboratory Service Board, London, England. Bacterial envelope preparations. Whole bacterial envelopes were prepared by ultrasonication and differential cen-
Corresponding author. 1774
VOL. 58, 1990
CROSS-REACTIVITY OF BACTERIAL ANTIGEN AND HLA
TABLE 1. HLA phenotype of cell lines used in testing monoclonal antibodies
HOM-2 Daudi K562 MM 1065 EL-4 (mouse)
EB-lb EA-6b a
b
TABLE 2. Specificity of monoclonal antibodies for cultured cell lines Specificity for the indicated celi line
MHC class I surface antigena
Cell line
1775
Cell line HLA-A
HLA-B
HLA-C
3
27
1
2,24 24
27,44
2,5 2
7 2
MHC, Major histocompatibility complex. Transfected mouse cell line (14).
trifugation as described previously (29). Total protein content was determined by the method of Lowry et al., modified for membrane-associated proteins (24). Production of monoclonal antibodies. BALB/c female mice were inoculated in multiple subcutaneous sites with 100 ,ug of total protein suspended in 0.1 ml of normal saline. Mice were rested for 1 month and then given a final intraperitoneal inoculum of 100 ,ug of total protein. The immunogen for all fusions was whole bacterial envelopes prepared from S. flexneri 2a isolated from a patient with Reiter's syndrome (SSU 6335). Splenocytes were fused with syngeneic plasmacytoma cells P3x63.Ag8.653) as described by Kohler and Milstein (19) and modified by Kennett (17) and Oi and Herzenberg (26), with polyethylene glycol 1500 (BoehringerMannheim Biochemicals, Indianapolis, Ind.) as the fusogen. Positive hybridomas were cloned by using the Epics CS flow cytometer equipped with an autoclone accessory. Antibodies were isotyped by radial immunodiffusion by using rabbit anti-mouse subclass-specific antisera (Binding Site, Inc., San Diego, Calif.). Ascites fluids were produced as previously described (15). Immunoglobulin M (IgM) was purified from ascites fluids by Sephadex G200 (Pharmacia Fine Chemicals, Piscataway, N.J.) chromatography followed by hypoosmotic precipitation by dialysis of IgM-containing fractions against 1 mM Tris hydrochloride (pH 7.5). Synthetic polypeptides. Peptides were synthesized in an automated peptide synthesizer (model 430A; Applied Biosystems, Inc., Foster City, Calif.). Peptide from the solidphase support was deprotected and released by treating the protected resin-attached peptide with anhydrous hydrofluoric acid containing 10% anisole for 2 h at 0°C. Peptide purity was monitored by reversed phase high-pressure liquid chromatography. Peptides were determined to be >90% pure. ELISA. Hybridomas were screened for antibody production by an enzyme-linked immunosorbent assay (EUSA) with 0.05 p.g total bacterial envelope protein applied to each well of 96-well flat-bottomed polystrene microdilution plates (Immulon I; Dynatech Laboratories, Inc., Chantilly, Va.). Hybridoma supernatants (100 1d) were pipetted into coated wells and incubated for 2 h at room temperature. Bound antibody was detected with a 1:1,000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (heavy and light) (Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.) with p-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, Mo.) as the chromogen. ELISAs with synthetic polypeptide were optimized to 10 ,ug/well of peptide bound to Dynatech Immulon II microdilution plates. The ELISAs were carried out as described above by using 100 1.l of a 2-,ug/ml solution of purified IgM suspended in 1% normal goat serum.
A7E2
HOM-2 Daudi K562 MM 1065 EL-4 EB-1 EA-6
C7E1O
E2B6
FlOE10
D1D8a
-
+
+
+
-
-
-
-
-
+
+
+
+
-
+ -
+ _
+ _
+ -
+
+
+
+
_ _
-
-
-
-
-
+
a Isotype control.
Flow cytometry. Reactivity of monoclonal antibodies toward native HLA molecules was assessed by indirect immunofluorescence with transformed human lymphoblastoid cell lines or transfected murine cells that express HLA surface antigen. Daudi and K562 cell lines were obtained from American Type Culture Collection, Rockville, Md. HOM-2 and MM cells (HLA-B27 positive) and an MMderived HLA-B27 negative mutant (1065) were provided by D. T. Y. Yu. The murine T-cell iymphoma line (EL-4) and EL-4-derived transfectants (EA-6, EB-1) were obtained from Victor H. Engelhard, University of Virginia School of Medicine, Charlottesville, Va. (14). All cells were maintained in RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and nonessential amino acids. Transfected cells were maintained in the same medium that also contained 250 ,ug of G418 (GIBCO Laboratories, Grand Island, N.Y.) per ml. Methods for fluorescent staining of live cells were performed as previously described (30) with a 1:5 to 1:10 dilution of hybridoma supernatant and a 1:10 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgM F(ab')2 fragments (Jackson). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis. Cultured lymphocytes (108) were washed three times by centrifugation for 5 min at 500 x g in 0.4 M NaCl-200 mM Tris hydrochloride (pH 7.4). Membrane proteins were extracted by resuspending the cell pellet in 1 ml of the same buffer containing 0.5% Nonidet P-40 (Pierce Chemical Co., Rockford, Ill.) and 1 mM phenylmethylsulfonyl fluoride (Sigma). After incubation for 30 min on ice, extracted cells were pelleted for 1 h at 40,000 x g. Supernatant samples containing 21 ,ul of membrane extract (40 to 50 pg of total protein) were examined by electrophoresis under reducing conditions by the method of Laemmli (22) with a 12.5% polyacrylamide resolving gel. Alternatively, pelleted bacterial envelopes were suspended in sample buffer, and 25-,ul samples (50 ,ug of total protein) were electrophoretically separated under the same conditions. Methods for transfer of proteins to nitrocellulose membranes were as described earlier (34). After blocking for 1 h with 1% bovine serum albumin (radioimmunoassay grade; Sigma) in 0.04 M Tris-0.5 M NaCl (pH 7.5), reactive bands were observed by using a 1:5 to 1:10 dilution of hybridoma supernatant and a 1:1,500 dilution of peroxidaseconjugated goat anti-mouse IgM. Blots were developed with a solution of 0.05% 4-chlor-1-naphthol ard 0.006% H202. RESULTS Antibody-secreting hybridomas were identified by ELISA with envelope proteins of S. flexneri 2a as the solid-phase
INFECT. IMMUN.
WILLIAMS AND RAYBOURNE
1776
U)
LUI LI.
0
LU
z
iq
Lu
I.
;j,i~
#
1-I LU
if. D1D8
LOGIo FLUORESCENCE INTENSITY FIG. 1. Immunofluorescent analysis of cultured lymphocytes and transfected murine cells with monoclonal antibodies. Cells were stained by an indirect method using hybridoma supernatants followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgM F(ab')2 fragments as described in Materials and Methods. HLA-cross-reactive antibodies (A7E2, FlOE10) are compared with the non-cross-reactive isotype control (D1D8).
reactant. Culture fluids with an A405 of >1.0 were then evaluated for specificity of binding to the cultured lympho-
cytes listed in Table 1. Of more than 600 antibacterial antibodies tested by flow microfluorometry, 4 were selected for further characterization, based on intensity of surface fluorescence observed when live cells were stained by an
indirect method. These four antibodies (designated A7E2, C7E10, E2B6, and FlOE10) were all of the IgM isotype. An additional antibacterial IgM monoclonal antibody, designated D1D8, was randomly chosen for use as an isotype control. Results of flow cytometric assays are summarized in Table
VOL. 58, 1990
CROSS-REACTIVITY OF BACTERIAL ANTIGEN AND HLA
1777
TABLE 3. Sequences of MHC class I-specific synthetic polypeptides (variable region) Amino acid sequence
Peptide 65
HLA-B*2705a HLA-B7 HLA-B40
70
E
T
N
T
Q Q
I
E
T
Q
I
I
C Y S
75
80
K
A
K
A
D
R
E
D
L
R
T
L
L
A
Q
A
Q Q
T
K
T
D
R
E
L
R
N
L
R
G
K
A
N
T
Q
T
Y
R
E
S S
L
R
N
L
R
G
R
a Formerly designated HLA-B27.1. Contains sequence required for B27.M2 binding (5).
2. HOM-2 cells, which are homozygous at the HLA-B locus, were strongly positive when stained with a saturating concentration of each of the four test monoclonal antibodies. By contrast, Daudi and K562 cells, which lack cell surface expression of class I molecules, were uniformly negative (Fig. 1). MM cells displayed a level of surface fluorescence comparable to that of HOM-2 cells. The mutant cell line 1065, which was derived from the HLA-B27-positive cell line MM but does not express for HLA-B locus antigens, was also tested. Although the antibodies stain MM cells more brightly, the 1065 cells demonstrated observable levels of fluorescence staining (Fig. 1). The transfected cell line EB-1, which expresses surface HLA-B7, was also positive with all four test monoclonal antibodies; however, cells transfected with and expressing HLA-A2 (EA-6) and the parental cell line (EL-4) were negative (Fig. 1). To characterize the molecular basis for antibody activity, synthetic polypeptides were produced with an amino acid sequence identical to the variable region-spanning residues 63 through 83 of the HLA-B*2705, HLA-B7, and HLA-B40 heavy chains (Table 3). Monoclonal antibodies were tested for antipeptide activity by ELISA. All antibodies were positive with each of the synthetic polypeptides, although preferential binding was observed with the HLA-B*2705 peptide and the homologous antigen (S. flexneri 6335 enve-
lope) (Fig. 2). Antibodies were nonreactive with two synthetic peptides of unrelated sequence, which were included as negative controls (data not shown). Membrane proteins of the HLA-B27-positive cell line MM were extracted with detergent, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose for Western blot analysis with the HLAreactive monoclonal antibody (Fig. 3). All monoclonal antibodies bound to a protein with a molecular mass of approximately 44 kDa, which is identical to that of the MHC class I heavy chain. A protein of similar molecular mass was identified by the B27-specific IgM monoclonal antibody B27.M2, which was included as a positive control. Other isotype controls were nonreactive by Western blotting. When tested by immunoblotting with electrophoretically resolved S. flexneri 2a membrane proteins, each of the four HLA-reactive monoclonal antibodies gave an identical pattern of reactivity. A major reactive protein was observed at 36 kDa, and a less prominent band was seen at 19 kDa. A I 1
B
C
D
E
F
G
6
84 58
SPECIFICITY OF ANTI-BACTERIAL ANTIBODIES
48.5
(Antibody Concentration * 2 ug/ml)
O.D. 406 nm
36.5
1.5 26.6
0.5
A7E2
W
C7E1O
HLA-B27 peptide HLA-B40 peptide
E2B6
FlOE10
D1D8
HLA-B7 peptide S. flexneri 6335
FIG. 2. Reactivity by ELISA of antibacterial monoclonal antibodies with HLA-B synthetic polypeptides and S. flexneri envelope proteins. Peptides were synthesized according to the sequence of their respective variable regions (amino acids 63 through 83). Assays (described in Materials and Methods) used monoclonal antibodies purified from ascites fluids. Cell-reactive monoclonal antibodies were positive against all three synthetic polypeptides, although stronger reactivity was observed with HLA-B27 peptide and bacterial envelope proteins. Control antibacterial monoclonal antibodies (D1D8 isotype control) were negative against synthetic peptides.
FIG. 3. Western blot analysis of detergent-extracted MM cells (HLA-B27 positive) with monoclonal antibodies. Cell membrane proteins were extracted with Nonidet P-40 and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis as described in Materials and Methods. Antibacterial monoclonal antibodies (lanes B through E) and the HLA-B27-specific monoclonal antibodies, B27.M2 (lane F), reacted with a protein with a molecular mass consistent with that of the major histocompatibility complex class I H chain (44 kDa). Lanes: A, Coomassie blue-stained membrane extract; B, A7E2; C, C7E10; D, E2B6; E, FlOE10; F, B27.M2; G, D1D8 (isotype control).
1778
INFECT. IMMUN.
WILLIAMS AND RAYBOURNE A
3
C
D
E
F
84
588
.....
48.5
*_9MOW-' 4f,W
AII
26.6
FIG. 4. Western blot analysis of S. flexneri envelope proteins with monoclonal antibodies. All of the HLA-reactive monoclonal antibodies reacted with proteins of 36 and 19 kDa; the 36-kDa protein was the most prominent. Control antibacterial monoclonal antibodies gave a different pattern of reactivity. Lanes: A, Coomassie blue-stained envelope proteins; B, A7E2; C, C7E10; D, E2B6; E, FlOE10; F, D1D8 (isotype control).
Neither protein reacted with antibody D1D8, which was included as an isotype control (Fig. 4). To determine whether the cross-reactive epitope was unique to this isolate of S. flexneri, envelope proteins were prepared from a collection of S. flexneri strains representing most serotypes and from a group of related bacteria, some of which are also epidemiologically associated with arthritic disease. These preparations were standardized for protein concentration and tested by ELISA with the monoclonal antibody. Positive activity against bacterial strains was observed with the HLA-reactive monoclonal antibodies (Table 4). The isotype control was specific for S. flexneri 2a. Although the reaction of antibody C7E10 was weaker than that of the remaining three antibodies, no preferential binding was seen with any strains tested.
DISCUSSION Despite the epidemiologic evidence associating gram-negative members of the family Enterobacteriaceae with the seronegative spondyloarthropathies, the mechanism by which bacterial factors can initiate and perpetuate these diseases remains hypothetical. Furthermore, in contemplating pathogenic mechanisms, equal consideration must be given to genetic factors, such as the presence of the class I histocompatibility antigen HLA-B27, which undoubtedly predisposes individuals to postinfectious arthritic sequelae (6). The results presented in this report demonstrate that monoclonal antibodies prepared against an arthritogenic organism also recognize epitopes present on class I histocompatibility antigen. This supports the hypothesis that the link between environmental and genetic etiologic factors is a common immunodeterminant and that this molecular mimicry could be of pathologic significance in the induction of chronic arthritic disease. The four cross-reactive monoclonal antibodies were initially identified by using cultured cell lines in an indirect microfluorometric assay. Although previous investigators have used a microcytotoxicity assay to determine HLA
specificity of their monoclonal antibodies (20), we tried to avoid any potential problems associated with complementbinding capabilities of the monoclonal antibodies. The four monoclonal antibodies had similar patterns of reactivity against the cultured cell lines. Antibodies were completely negative against cells lacking MHC class I antigens (Daudi, K562, EL-4) and against murine lymphoid cells transfected with and expressing only HLA-A2 (EA-6). Monoclonal antibodies were not, however, singularly positive with HLAB27-positive cells; they were equally reactive with transfectants expressing only HLA-B7 (EB-1) and nominally reactive with cells lacking HLA-B antigen but expressing HLA-A24 and HLA-C2 (1065). This low degree of allelic specificity indicates that the epitope recognized by these antibodies consists of nonpolymorphic residues shared by several HLA class I antigens. We can also conclude that this epitope differs from that recognized by B27.M2, which is specific for B27 and Bw47 (5). Although it appears that our monoclonal antibodies bind preferentially with B-locus antigens, fluorescence intensity between cultured lymphocytes may not be directly comparable, because the surface density of HLA antigen may vary from one cell line to another. In Western blot analyses against detergent-extracted MM (HLA-B27-positive) cells, all four cross-reactive monoclonal antibodies bound to a single band at an approximate molecular mass of 44 kDa. This molecular mass corresponds to that previously reported for the MHC class I heavy chain and is identical to that observed when monoclonal antibody B27.M2 is subjected to immunoblot analysis. Based on ELISA results with synthetic polypeptides corresponding to amino acids 63 through 83 of the class I heavy chain, we determined that the epitope recognized by our monoclonal antibodies was present in this segment. Previous investigators have shown that this polymorphic sequence also contains the epitope responsible for specific binding of B27.M2 (5) and recognition structures for allospecific cytotoxic T lymphocytes (2, 41). Although this segment would appear critical in defining allospecificity of the HLA-B27 molecule, significant structural homology exists between the HLA-B27 variable region and the corresponding segment of nonHLA-B27 class I molecules (32). Several nonpolymorphic epitopes within the variable region have been identified by using monoclonal antibodies generated against lymphocytic cells (1, 11, 27, 28, 31). The existence of such epitopes would explain the reactivity of our four monoclonal antibodies with non-HLA-B27 class I antigen and in particular with the HLA-B7 and HLA-B40 synthetic polypeptides, which were also derived from their respective variable regions. The presence of such epitopes may also explain the high incidence of HLA-B7 CREG (cross-reactive group) antigens in HLA-B27-negative spondyloarthritic patients (4, 18). Using S. flexneri envelope proteins, we observed qualitatively identical patterns of immunoblot reactivity for the four monoclonal antibodies. The reactive epitope was present on 36- and 19-kDa antigen of S. flexneri, but was not restricted to this species of the family Enterobacteriaceae. Strong ELISA activity was observed with all strains tested, even with species generally considered nonarthritogenic (e.g., E. coli). Despite the presence of such antigens, additional bacterial virulence mechanisms (such as the ability to invade host cells and survive intracellularly) are also characteristic of species with arthritogenic potential. Indeed, the ability of an organism to establish infection is a prerequisite for host exposure to and immune system recognition of cross-reactive antigens. The occurrence of a cross-reactive epitope that is present on many organisms is consistent with the
VOL. 58, 1990
CROSS-REACTIVITY OF BACTERIAL ANTIGEN AND HLA
1779
TABLE 4. Reactivity of monoclonal antibodies with enterobacterial envelope proteins
A405
Bacterial strain A7E2
C7E1O
E2B6
FlOE10
D1D8I
la 1235-66 la 1921-71 lb 6115b lb 4343-70 lb 5612-73 2a 6335b 2a 947b 2a 2747-71 2a 29903 2b 12022 2b 1676 2b 9768 3 2146-66 3 2783-71 4a 6603-63 4a 1862-71 4b 880-69 4b 1242-70 5 1170-74 5 5103-82 6 2924-71 6 796-83
1.845 1.484 1.801 1.223 1.328 1.909 1.795 1.505 0.920 1.639 1.437 1.362 1.430 1.557 1.570 1.760 1.337 1.002 1.164 1.166 1.163 1.132
0.652 0.597 0.411 0.454 0.478 0.517 0.406 0.574 0.269 0.470 0.430 0.297 0.486 0.568 0.531 0.454 0.445 0.347 0.406 0.441 0.428 0.429
1.888 1.604 1.115 1.172 1.343 1.527 1.679 1.508 1.070 1.316 1.410 1.023 1.564 1.586 1.484 1.517 1.426 1.036 1.038 1.149 0.996 1.030
1.285 1.384 1.265 1.012 1.439 1.420 1.397 1.167 0.653 1.496 1.024 1.011 1.115 1.163 1.193 1.296 1.907 1.276 1.804 1.496 1.332 1.532
0.086 0.057 0.056 0.050 0.048 1.836 1.947 1.735 1.389 0.075 0.054 0.049 0.051 0.052 0.049 0.070 0.072 0.058 0.048 0.063 0.055 0.059
Shigella sonnei 6425 7063
1.754 1.890
0.449 0.602
1.933 1.740
1.226 1.341
0.071 0.055
Salmonella heidelburg 7375b
1.204
0.434
1.342
1.204
0.085
Salmonella typhimurium 24b
1.690
0.511
1.872
1.226
0.078
Klebsiella pneumoniae K43
1.289
0.337
1.365
0.760
0.058
Escherichia coli 10908 3374
1.835 1.995
0.611 0.655
1.745 1.885
1.329 1.526
0.064 0.054
1.993 1.180
0.698 0.425
1.992 1.174
1.686 0.946
0.066 0.951
1.289 1.567
0.577 0.763
1.496 1.926
0.855 1.948
0.054 0.074
Shigella flexneri
Yersinia pseudotuberculosis 78 b
269b
Yersinia enterocolitica
A2b A6b
a Isotype control. b Isolated from a patient with Reiter's syndrome or reactive arthritis.
finding that many species of gram-negative bacteria are epidemiologically implicated in these diseases. Furthermore, many of the 36- to 38-kDa major outer membrane proteins of members of the family Enterobacteriaceae are conserved phylogenetically and considered common antigens (13), making them candidate recognition structures for our crossreactive monoclonal antibodies. A comparison of the relative mobility in polyacrylamide gels of the bacterial antigen recognized by B27.M2 indicates that our monoclonal antibodies have a different antigen specificity. The molecular specificity of our antibodies is currently under investigation. Further studies on humoral and cell-mediated responses of spondyloarthritic patients which parallel the course of their disease are required to determine the pathologic significance of the 36- and 19-kDa antigens. A vigorous antibody response against the causative organism is present in all patients after a gram-negative enteritis; however, those who
develop subsequent arthritis show higher and more persistent antibody titers in serum (35, 36, 39). Serum antibodies of the IgG, IgM, and IgA classes directed against Yersinia proteins of 35 to 36 kDa have been reported in patients with reactive arthritis after infection with Y. enterocolitica (12, 33, 40), but whether this antigen corresponds to the 36-kDa antigen recognized by our cross-reactive monoclonal antibody is yet to be determined. The availability of monoclonal antibodies will facilitate purification of this antigen for further studies with patient specimens. Of greater interest is the potential regulatory role of bacterially induced HLA class I-reactive antibodies on Tcell responses in a susceptible host. Several monoclonal anti-class I HLA antibodies, in the presence of adherent antigen-presenting cells, can inhibit mitogen- and antigeninduced T-cell proliferation (9, 10, 37). An antibody-mediated down-regulation of cellular responses to arthritogenic
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WILLIAMS AND RAYBOURNE
bacteria could facilitate persistence of bacteria in infected cells. Indeed, defective cellular immune functions have been reported in arthritic patients. Leino et al. (23) found that lymphoproliferative response to bacterial antigen was depressed in the peripheral blood mononuclear cells of patients who developed post-Yersinia reactive arthritis as compared with those who recovered from yersiniosis uneventfully. The impaired response was not organism specific, since significant inhibition was obtained after stimulation with either Yersinia or E. coli antigen. Similarly, peripheral blood mononuclear cells from patients with reactive arthritis after Salmonella typhimurium infection showed an impaired lymphoproliferative response to in vitro stimulation with the causative organism (16). Hypothetically, HLA-B27-reactive antibodies could be induced by a common bacterial antigen in all infected patients, but their negative regulatory effects would be expressed only in patients who are HLA-B27 positive or whose class I antigens contain the cross-reactive
epitope. In conclusion, we identified 36- and 19-kDa bacterial envelope proteins that share cross-reactive epitope(s) with class I histocompatibility molecules. Although the mechanism by which these or other bacterial antigens precipitate joint disease remains unknown, the existence of shared immunodeterminants lends support to the concept of molecular mimicry in disease induction.
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