Clin. exp. Immunol. (1992) 89, 230-238
Immunoglobulin heavy chain variable region gene utilization by B cell hybridomas derived from rheumatoid synovial tissue C. M. S. BROWN, C. LONGHURST, G. HAYNES*, C. PLATER-ZYBERK, A. MALCOLM* & R. N. MAINI Kennedy Institute of Rheumatology and *Charing Cross and Westminster Medical School, Charing Cross Hospital, London, UK
(Acceptedfor publication 5 May 1992)
SUMMARY Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects synovial joints. Activated B lymphocytes and plasma cells are present in the synovial tissue and are thought to contribute to the immunopathology of the rheumatoid joint. To investigate rheumatoid synovial B lymphocytes, we have generated B cell hybridomas from synovial tissue of an RA patient. Here we describe the immunoglobulin VH gene repertoire of eight IgM- and 10 IgG-secreting synovial-derived hybridomas. The VH4 gene family is highly represented (38 5%) in this panel of hybridomas compared with the frequency of VH4 gene expression in circulating B lymphocytes reported previously (19-22%) and with the VH4 gene frequency we observed in a panel of hybridomas derived in the same manner from the spleen and tonsil of normal individuals (19%). The increased frequency of VH4 gene expression was not due to the expansion of a single B cell clone in vivo as none of these hybridomas was clonally related. Two synovial-derived hybridomas secreted autoantibodies; one (VH3 +) secreted an IgM-rheumatoid factor (RF) and the other (VH4+) secreted IgM with polyreactive binding to cytoskeletal proteins and cardiolipin. The antibodies secreted by the remaining synovialderived hybridomas were not reactive with the autoantigens tested. The VH gene usage in a proportion (5/17) of synovial-derived hybridomas that expressed CD5 antigen provided preliminary evidence that CD5 + B cells in RA synovium have a similar increase of VH4 gene expression reported for CD5+ B cells from normal individuals and patients with chronic lymphocytic leukaemia.
Keywords immunoglobulin variable genes B cell hybridomas rheumatoid arthritis synovium antibodies secreted locally in the synovium to be studied in INTRODUCTION with the genes that encode them. In this report we parallel Rheumatoid arthritis (RA) is a disease of unknown etiology examine the immunoglobulin VH genes utilized by a panel of 10 which is associated with autoimmune manifestations. The IgG- and eight IgM-secreting hybridomas derived from a primary lesion occurs in the synovial joints and is characterized with seropositive RA and compare the pattern of patient by chronic inflammation and immunological reactivity in the with that observed in normal splenic and tonsillar B expression synovial tissue. Activated B cells and plasma cells secrete documented in the literature for circulating B cells that cells and immunoglobulin, including rheumatoid factors (RF) and anti[7,8]. collagen antibodies, into the synovial tissues and joint space Immunoglobulin H chain V-region genes are assembled [1,2]. These autoantibodies may exert pathogenic effects via from three gene segments, VH, D and JH, that exist as multiple [3]. There fixation complement and formation immune.complex hr n imun oplmn iain[ are approximately 100-200 VH germline DNA. copies DNA. There is also emerging evidence that RF-secreting B cells can capture Thereare approximtely copes iniermlin n[] hc r lsiidit i Hfmle gee.rvee immune complexes and present these theseantigens to T cells [,5] based on the nucleotide homology between individual genes t Thism ionthe roeo [10-12]. The factors that govern the selection of particular VH synovial immune response. Thereforei understanding the rolers genes during immunoglobulin gene rearrangement appear to be synovial B cells in RA is important in determining the factors reuaeiin part, ytm at by ytemtrt the immune system, the maturity offteimn regulated, inovdiheptoeei of thi dies. use B adult cells, with when B fetal because compared cells, taizeBel i toimmortalize B cells from synovial Our approach has been to restricted sets of VH genes [13-16]. Some murine autoantibodyo-admutlzto fV sertn B.el.loehbi tissue by generating B cell hybridomas [6]. This has enabled
cope.omto
100-200lVH
iomple anvved perpgetationfcells echanismumaynbe Thisynovialnismmun response.lThereforehersetuan
apvolvedintheproachohasnbeen
genes [17]. However, this has not been clearly established in human autoimmune disease. We have determined the utilization of the six human VH gene families in a panel of synovial-derived
Correspondence: Professor R. N. Maini, Clinical Immunology Division, Kennedy Institute of Rheumatology, 6 Bute Gardens, Hammersmith, London W6 7DW, UK.
230
Immunoglobulin VH gene utilization in RA synovium hybridomas as a preliminary approach to studying the VH gene repertoire of B cells in RA. In addition, we have been able to examine the VH genes used by a proportion of the hybridomas which expressed the CD5 antigen. CD5+ B cells are associated with autoimmune disease because they commonly secrete autoantibodies and are increased in number in autoimmune mouse strains [18-20] and in some human autoimmune diseases, including RA [21-23]. The VH gene repertoire of CD5 + B cells in chronic lymphocytic leukaemia (CLL), where they are the malignant B cell population, is biased towards an increased expression of the VH4, VH5 and VH6 gene families [24-26]. There are also preliminary data indicating that the VH gene repertoi arof CD5 + B cells in normal individuals shares siia similar biases [27]. The expression of CD5 iniiul shre antigen on the surface of a proportion of the synovial-derived antigen on has enabled uuss topporetemione theVH syenoutilized determine the hybridomas genes utilize by these cells in rheumatoid synovial tissue.
biss.7.Teexrsino
theasurfabledf
MATERIALS AND METHODS
231
Immunoglobulin C region DNA probes for y-H chain [34] and p1-H chain [35] were generously provided by Dr M-P. Lefranc, University of Montpellier, Montpellier, France, and Dr T. Rabbitts, Laboratory of Molecular Biology, Cambridge, UK, respectively. Probes were labelled with [a-32P]dCTP using a random-priming oligo-labelling kit (Pharmacia/LKB, Milton Keynes, UK) based on the random priming method described by Feinberg & Vogelstein [36]. Northern blotting Total RNA (20 pg) was separated on a 1% (w/v) agarose gel in R (20 u37t at 1% was tansfered 2o2 2 2 M formamide [37] at 30 V overnight. RNA was transferred to Hybond-N nylon membrane (Amersham, Amersham, UK) and covalently linked to the membrane by exposing to u.v. light. Membranes were prehybridized in a solution containing 5 x ouincnann ebaeaeepeyrdzdi Denhardt's (1 x Denhardt's 0 05% (w/v) 05% (w/v) 0 05%is 0(w/v) bovineFicoll, serum albumin to
polyvinylpyrollidone,
(BSA)), 50% (v/v) formamide,
5 x SSC, 0-1% (w/v) SDS and 150 jug/ml yeast tRNA for 1 h at 420C and hybridized in an identical solution containing the labelled probe overnight at 420C. Membranes were washed twice at low stringency (2 x SSC, 0 1% (w/v) SDS; 15 min at room temperature) followed by two high stringency washes (0 1 x SSC, 01 % (w/v) SDS; 30 min at 55&C) and exposed to X-ray film (Fuji RX, GRI, Dunmow, UK) for 3-5 days at - 70'C with intensifying screens. Each membrane was probed sequentially with the six VH familyspecific probes and with the appropriate C region probe. Between successive hybridizations, membranes were stipped of bound probe by a 1-2 h wash at 650C in 5 mm Tris-HCl (pH 8-0), 2 mM Na2EDTA, 0 1 x Denhardt's. RNA size markers were included on each gel for comparison.
B cell hybridomas Ten IgG- and eight IgM-secreting B cell hybridomas derived from the synovium of a patient with RA (patient 1) were used in this study. Patient 1 was a 59-year-old woman with classical erosive seropositive RA who satisfied the ARA criteria [28]. The duration of her disease was 6 years. At the time of surgery her medication included piroxicam, aspirin and salazopyrin. Her HLA type was A2,-; B44,60; DR4,-; DRw53,-; DQ3,-. Synovial lymphocytes were fused with the heteromyeloma cell fusion partner, SPAZ-4 [29] and were not transformed or deliberately activated in vitro before fusion. The construction of these hybridomas has been described previously [6]. B cell hybridomas were also derived using the same fusion surfacewere stained CD5 for expression Hybridomas CD5 expression using a direct procedure from the mononuclear cells extracted from the spleen immunofluorescent staining technique. Cells (3-5 x 150) were of a 37-year-old patient undergoing surgery for the removal of distributed in 12 x 75 mm tubes and washed twice with PBS stomach carcinoma and from the tonsils of a 3-year-old child containing 2% (v/v) bovine serum and 01 % (w/v) sodium azide undergoing tonsilectomy. These hybridomas were obtained (washing buffer). The cell pellets were incubated with 5 yl PEfrom four independent fusions as two fusions were carried out conjugated MoAb (1:5 (IgG2a subclass) 15Rmm (Ti RD1,onlie. Coulter conics, CD5 LutonU dution) for each fr~mourndepndetfusonsstwfuslnswrecarleout of the splenic and tonsillar tissues. In one of each of staicel susons wer washedltice in washing bfer, these paired fusions, the cells were incubated for 3 days before c fusion with Staphylococcus aureus Cowan strain (SAC) (Calbioresuspended in. 200 p1 buffer and applied through the flow chem, Novabiochem, Nottingham, UK) (20 pg/ml) and pokecytometer (FACScan, Becton Dickinson, Oxford, UK). Results weed mitogen (PWM) (Sigma, Poole, UK) (10 pg/ml). The analysed using the Consort 30 software (FACScan, Becton used fortheotherwofusonsweewere mononuclear cells usedcells for the other two fusions were used Dikno)Moboftesmioypbuirlvatpcfickityower inlue in tea staining to determinevt limit directly and not incubated with mitogens. Twenty one spleenficity were included in each staining to determine the limits of and onsl-drive wee ued iinthisstuy. and onsi-deivedhybrdoms thi stdy. hey hyridmaswereuse negativity. The cell line (SPAZ-4) used as fusion partner to gnerate these Theselhybidm was aso ed foriCD presi. comprised seven IgM-, 11 IgG- and three IgA-secreting hybrihybridomas was also tested for CD5 expression. domas. domas.
~~~~~~~~~~~~~generate
RNA extraction
Synovial-derived hybridoma cells were lysed in 4 M guanidium isothiocyanate [30] and total RNA purified by caesium chloride gradient centrifugation [31]. Total RNA was purifed from spleen- and tonsil-derived hybridomas using a more rapid single-step method using a guanidinium thiocyanate-phenolchloroform extraction according to the procedure described by Chomczynski & Sacchi [32]. Gene probes VH gene family-specific probes were generously provided by Dr Fred Alt, Columbia University, NY (VHI, VH3, VF,4, VHS and VH6) [12] and by Dr T. Honjo, Kyoto, Japan (VH2) [33].
Screening hybridoma culture SNfor reactivity with autoantigens Undiluted hybridoma culture supernatants (SN) were screened for reactivity with the following antigens by ELISA; human collagen type II (prepared in the department according to the method described [38]), bovine muscle G-actin (Sigma), rabbit skeletal muscle myosin (a generous gift from Dr F. Walsh, Cambridge, UK), bovine lens vimentin (ICN ImmunoBiologicals, High Wycombe, UK), bovine heart cardiolipin (Sigma), calf thymus single-strand (ss)DNA (Sigma), tetanus toxoid (Tetanus vaccine BP, Wellcome, Dartford, UK) and influenza A and B haemagglutinin and neuraminidase antigens (Fluvirin vaccine, Evans Medical Co., Greenford, UK). Standard ELISA techniques were used. Antigens were coated to microtitre plates (Falcon, Becton Dickinson, Oxford,
C. M. S. Brown et al.
232 to
m cat
CM
_
VH] I.
N
ca
1i-o C
a
6
2
binding to the microtitre plate was blocked by incubation with casein (BDH, Poole, UK) (2'%, (w/v) in PBS). PBS containing 001(X Tween-20 was used for all washing procedures. Bound C4l I I I human antibody was revealed by incubation with alkalinephosphatase conjugated goat anti-human IgG or IgM (Sigma) (diluted 1:1000 in PBS containing 0 01% Tween-20) and detected by incubation in p-nitrophenol substrate (Sigma). 5 @ . In some instances there were modifications to this technique. Anti-collagen and anti-cardiolipin ELISA were carried out at room temperature. Cardiolipin was coated to microtitre plates by dissolving in ethanol and air-drying onto the plate. Tween-20 -_.was not used in the washing or dilution buffers for anti' ELISA. All ELISA results were compared with the binding of the .. .... _. . .- ... _supernatants to microtitre plates that had been incubated with casein, but not coated with antigen. Indirect immunofluorescent (IIF) staining techniques were used to measure the reactivity of undiluted culture SN with nuclear and cytoskeletal components of HEp-2 cells and 3T3 fibroblasts respectively and with double-strand DNA in Crithi_- ...... '- _ dia lucillae on multitest slides (Biodiagnostics, Malvern, UK). Standard IIF staining techniques were used with 20-min incubations at room temperature. A FITC-conjugated goat anti-human immunoglobulin (diluted 1:50 in PBS) was used to reveal human antibody bound to the substrate. IgM-RF and IgG-RF in culture SN has been measured previously [6]. t
0 4 4: 4 I l II I I
*
s . ~~~~~~~~~~~~cardiolipin
VH2
VH3
VN 4
*
Vat 5 t
SRESULTS
VHG6
Fig. 1. Total RNA extracted from each hybridoma was probed sequentially with each of the DNA probes specific for the human VH gene families 1-6 and for IgM and IgG C regions (Cp and Cy). Hybridization with the C region probes confirmed the presence of immunoglobulin messenger RNA. The VH probes did not crosshybridize when the filters were washed at high stringency. A representative panel of 10 hybridomas is shown here. UK) at 10 pg/ml in PBS by incubation at 40C overnight. All subsequent incubations were for 1 h at 370C. Non-specific
VH gene family usage in synovial-derived B cell hybridomas
Hybridizing bands of 2-4 and 2-2 kb with C region probes (p and y respectively) ascertained the presence of immunoglobulin message in the total RNA extracted from each hybridoma. RNA from each hybridoma hybridized with only one of the six VH gene probes (Fig. 1). The relative expression of each VH gene family in IgM- and IgG-secreting synovial-derived hybridomas is depicted in Table I . VH genes from families VH 1, VH2, VH3 and VH4 were represented in the 18 hybridomas, VH genes from VHS and VH6 families were not expressed.
Table 1. VH gene usage in IgM-, IgG- and IgA- secreting hybridomas
Hybridomas derived from non-rheumatoid spleen and tonsil
Hybridomas derived from rheumatoid synovium VH gene
family I 2
3 4 5 6
IgM-secreting (n=8) 1 (1250%) 1 (125-%) 3 (37.50/) 3 0 0
(37*5%")
IgG-secreting (n= 10)
IgM-secreting (n=7)
IgG-secreting (n= 11)
IgA-secreting (n=3)
2 (20%,)
1 (143'%)
3 (273%/o)
0
0
0 5 (71
0 5 (45
0 1
4 (40%Y) 4 0 0
(40%.)
4'%)
0 1(143%.) 0
50%)
2 (18 2°%) 1(91-%) 0
(33 3%) 2 (66-7%)
0 0
All hybridomas were derived by fusing human mononuclear cells with a heteromyeloma cell line (SPAZ-4). VH gene family expression was determined by Northern blotting using DNA probes specific for each of the six human VH gene families.
Immunoglobulin VH gene utilization in RA synovium
233
Table 2. The frequency of VH gene expression in a panel of hybridomas derived from rheumatoid synovial tissue and non-rheumatoid spleen and tonsil, compared with the frequency of VH family expression observed in the peripheral blood of normal individuals
VH gene expression Hybridomas
Peripheral blood
VH gene family
Size in the genome (predicted frequency of expression, %)
Family
Rheumatoid synovial-derived hybridomas (n = 18)
hybridomas (n=21) 4 (19%) 0 11 (52 5%) 4 (19%) 2 (9-5%Yo)
3 (16-7%) 1 (56%) 7 (38-9%) 7 (38-9%)
20-25 (33) 5-10(11) 25-30 (40) 6-10 (11) 2-3 (4) 1 (1)
1 2 3 4 5 6
Non-rheumatoid spleen- and tonsil-derived
0 0
0
PBMC
EBV-lines
EBV-lines
By in situ hybridization [7]
by Northern blotting [8]
11% 3%
12% 0%
52% 19% 10% 5%
56%
15%
Immunoglobulin heavy chain variable region gene utilization by B cell hybridomas derived from rheumatoid synovial tissue C. M. S. BROWN, C. LONGHURST, G. HAYNES*, C. PLATER-ZYBERK, A. MALCOLM* & R. N. MAINI Kennedy Institute of Rheumatology and *Charing Cross and Westminster Medical School, Charing Cross Hospital, London, UK
(Acceptedfor publication 5 May 1992)
SUMMARY Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects synovial joints. Activated B lymphocytes and plasma cells are present in the synovial tissue and are thought to contribute to the immunopathology of the rheumatoid joint. To investigate rheumatoid synovial B lymphocytes, we have generated B cell hybridomas from synovial tissue of an RA patient. Here we describe the immunoglobulin VH gene repertoire of eight IgM- and 10 IgG-secreting synovial-derived hybridomas. The VH4 gene family is highly represented (38 5%) in this panel of hybridomas compared with the frequency of VH4 gene expression in circulating B lymphocytes reported previously (19-22%) and with the VH4 gene frequency we observed in a panel of hybridomas derived in the same manner from the spleen and tonsil of normal individuals (19%). The increased frequency of VH4 gene expression was not due to the expansion of a single B cell clone in vivo as none of these hybridomas was clonally related. Two synovial-derived hybridomas secreted autoantibodies; one (VH3 +) secreted an IgM-rheumatoid factor (RF) and the other (VH4+) secreted IgM with polyreactive binding to cytoskeletal proteins and cardiolipin. The antibodies secreted by the remaining synovialderived hybridomas were not reactive with the autoantigens tested. The VH gene usage in a proportion (5/17) of synovial-derived hybridomas that expressed CD5 antigen provided preliminary evidence that CD5 + B cells in RA synovium have a similar increase of VH4 gene expression reported for CD5+ B cells from normal individuals and patients with chronic lymphocytic leukaemia.
Keywords immunoglobulin variable genes B cell hybridomas rheumatoid arthritis synovium antibodies secreted locally in the synovium to be studied in INTRODUCTION with the genes that encode them. In this report we parallel Rheumatoid arthritis (RA) is a disease of unknown etiology examine the immunoglobulin VH genes utilized by a panel of 10 which is associated with autoimmune manifestations. The IgG- and eight IgM-secreting hybridomas derived from a primary lesion occurs in the synovial joints and is characterized with seropositive RA and compare the pattern of patient by chronic inflammation and immunological reactivity in the with that observed in normal splenic and tonsillar B expression synovial tissue. Activated B cells and plasma cells secrete documented in the literature for circulating B cells that cells and immunoglobulin, including rheumatoid factors (RF) and anti[7,8]. collagen antibodies, into the synovial tissues and joint space Immunoglobulin H chain V-region genes are assembled [1,2]. These autoantibodies may exert pathogenic effects via from three gene segments, VH, D and JH, that exist as multiple [3]. There fixation complement and formation immune.complex hr n imun oplmn iain[ are approximately 100-200 VH germline DNA. copies DNA. There is also emerging evidence that RF-secreting B cells can capture Thereare approximtely copes iniermlin n[] hc r lsiidit i Hfmle gee.rvee immune complexes and present these theseantigens to T cells [,5] based on the nucleotide homology between individual genes t Thism ionthe roeo [10-12]. The factors that govern the selection of particular VH synovial immune response. Thereforei understanding the rolers genes during immunoglobulin gene rearrangement appear to be synovial B cells in RA is important in determining the factors reuaeiin part, ytm at by ytemtrt the immune system, the maturity offteimn regulated, inovdiheptoeei of thi dies. use B adult cells, with when B fetal because compared cells, taizeBel i toimmortalize B cells from synovial Our approach has been to restricted sets of VH genes [13-16]. Some murine autoantibodyo-admutlzto fV sertn B.el.loehbi tissue by generating B cell hybridomas [6]. This has enabled
cope.omto
100-200lVH
iomple anvved perpgetationfcells echanismumaynbe Thisynovialnismmun response.lThereforehersetuan
apvolvedintheproachohasnbeen
genes [17]. However, this has not been clearly established in human autoimmune disease. We have determined the utilization of the six human VH gene families in a panel of synovial-derived
Correspondence: Professor R. N. Maini, Clinical Immunology Division, Kennedy Institute of Rheumatology, 6 Bute Gardens, Hammersmith, London W6 7DW, UK.
230
Immunoglobulin VH gene utilization in RA synovium hybridomas as a preliminary approach to studying the VH gene repertoire of B cells in RA. In addition, we have been able to examine the VH genes used by a proportion of the hybridomas which expressed the CD5 antigen. CD5+ B cells are associated with autoimmune disease because they commonly secrete autoantibodies and are increased in number in autoimmune mouse strains [18-20] and in some human autoimmune diseases, including RA [21-23]. The VH gene repertoire of CD5 + B cells in chronic lymphocytic leukaemia (CLL), where they are the malignant B cell population, is biased towards an increased expression of the VH4, VH5 and VH6 gene families [24-26]. There are also preliminary data indicating that the VH gene repertoi arof CD5 + B cells in normal individuals shares siia similar biases [27]. The expression of CD5 iniiul shre antigen on the surface of a proportion of the synovial-derived antigen on has enabled uuss topporetemione theVH syenoutilized determine the hybridomas genes utilize by these cells in rheumatoid synovial tissue.
biss.7.Teexrsino
theasurfabledf
MATERIALS AND METHODS
231
Immunoglobulin C region DNA probes for y-H chain [34] and p1-H chain [35] were generously provided by Dr M-P. Lefranc, University of Montpellier, Montpellier, France, and Dr T. Rabbitts, Laboratory of Molecular Biology, Cambridge, UK, respectively. Probes were labelled with [a-32P]dCTP using a random-priming oligo-labelling kit (Pharmacia/LKB, Milton Keynes, UK) based on the random priming method described by Feinberg & Vogelstein [36]. Northern blotting Total RNA (20 pg) was separated on a 1% (w/v) agarose gel in R (20 u37t at 1% was tansfered 2o2 2 2 M formamide [37] at 30 V overnight. RNA was transferred to Hybond-N nylon membrane (Amersham, Amersham, UK) and covalently linked to the membrane by exposing to u.v. light. Membranes were prehybridized in a solution containing 5 x ouincnann ebaeaeepeyrdzdi Denhardt's (1 x Denhardt's 0 05% (w/v) 05% (w/v) 0 05%is 0(w/v) bovineFicoll, serum albumin to
polyvinylpyrollidone,
(BSA)), 50% (v/v) formamide,
5 x SSC, 0-1% (w/v) SDS and 150 jug/ml yeast tRNA for 1 h at 420C and hybridized in an identical solution containing the labelled probe overnight at 420C. Membranes were washed twice at low stringency (2 x SSC, 0 1% (w/v) SDS; 15 min at room temperature) followed by two high stringency washes (0 1 x SSC, 01 % (w/v) SDS; 30 min at 55&C) and exposed to X-ray film (Fuji RX, GRI, Dunmow, UK) for 3-5 days at - 70'C with intensifying screens. Each membrane was probed sequentially with the six VH familyspecific probes and with the appropriate C region probe. Between successive hybridizations, membranes were stipped of bound probe by a 1-2 h wash at 650C in 5 mm Tris-HCl (pH 8-0), 2 mM Na2EDTA, 0 1 x Denhardt's. RNA size markers were included on each gel for comparison.
B cell hybridomas Ten IgG- and eight IgM-secreting B cell hybridomas derived from the synovium of a patient with RA (patient 1) were used in this study. Patient 1 was a 59-year-old woman with classical erosive seropositive RA who satisfied the ARA criteria [28]. The duration of her disease was 6 years. At the time of surgery her medication included piroxicam, aspirin and salazopyrin. Her HLA type was A2,-; B44,60; DR4,-; DRw53,-; DQ3,-. Synovial lymphocytes were fused with the heteromyeloma cell fusion partner, SPAZ-4 [29] and were not transformed or deliberately activated in vitro before fusion. The construction of these hybridomas has been described previously [6]. B cell hybridomas were also derived using the same fusion surfacewere stained CD5 for expression Hybridomas CD5 expression using a direct procedure from the mononuclear cells extracted from the spleen immunofluorescent staining technique. Cells (3-5 x 150) were of a 37-year-old patient undergoing surgery for the removal of distributed in 12 x 75 mm tubes and washed twice with PBS stomach carcinoma and from the tonsils of a 3-year-old child containing 2% (v/v) bovine serum and 01 % (w/v) sodium azide undergoing tonsilectomy. These hybridomas were obtained (washing buffer). The cell pellets were incubated with 5 yl PEfrom four independent fusions as two fusions were carried out conjugated MoAb (1:5 (IgG2a subclass) 15Rmm (Ti RD1,onlie. Coulter conics, CD5 LutonU dution) for each fr~mourndepndetfusonsstwfuslnswrecarleout of the splenic and tonsillar tissues. In one of each of staicel susons wer washedltice in washing bfer, these paired fusions, the cells were incubated for 3 days before c fusion with Staphylococcus aureus Cowan strain (SAC) (Calbioresuspended in. 200 p1 buffer and applied through the flow chem, Novabiochem, Nottingham, UK) (20 pg/ml) and pokecytometer (FACScan, Becton Dickinson, Oxford, UK). Results weed mitogen (PWM) (Sigma, Poole, UK) (10 pg/ml). The analysed using the Consort 30 software (FACScan, Becton used fortheotherwofusonsweewere mononuclear cells usedcells for the other two fusions were used Dikno)Moboftesmioypbuirlvatpcfickityower inlue in tea staining to determinevt limit directly and not incubated with mitogens. Twenty one spleenficity were included in each staining to determine the limits of and onsl-drive wee ued iinthisstuy. and onsi-deivedhybrdoms thi stdy. hey hyridmaswereuse negativity. The cell line (SPAZ-4) used as fusion partner to gnerate these Theselhybidm was aso ed foriCD presi. comprised seven IgM-, 11 IgG- and three IgA-secreting hybrihybridomas was also tested for CD5 expression. domas. domas.
~~~~~~~~~~~~~generate
RNA extraction
Synovial-derived hybridoma cells were lysed in 4 M guanidium isothiocyanate [30] and total RNA purified by caesium chloride gradient centrifugation [31]. Total RNA was purifed from spleen- and tonsil-derived hybridomas using a more rapid single-step method using a guanidinium thiocyanate-phenolchloroform extraction according to the procedure described by Chomczynski & Sacchi [32]. Gene probes VH gene family-specific probes were generously provided by Dr Fred Alt, Columbia University, NY (VHI, VH3, VF,4, VHS and VH6) [12] and by Dr T. Honjo, Kyoto, Japan (VH2) [33].
Screening hybridoma culture SNfor reactivity with autoantigens Undiluted hybridoma culture supernatants (SN) were screened for reactivity with the following antigens by ELISA; human collagen type II (prepared in the department according to the method described [38]), bovine muscle G-actin (Sigma), rabbit skeletal muscle myosin (a generous gift from Dr F. Walsh, Cambridge, UK), bovine lens vimentin (ICN ImmunoBiologicals, High Wycombe, UK), bovine heart cardiolipin (Sigma), calf thymus single-strand (ss)DNA (Sigma), tetanus toxoid (Tetanus vaccine BP, Wellcome, Dartford, UK) and influenza A and B haemagglutinin and neuraminidase antigens (Fluvirin vaccine, Evans Medical Co., Greenford, UK). Standard ELISA techniques were used. Antigens were coated to microtitre plates (Falcon, Becton Dickinson, Oxford,
C. M. S. Brown et al.
232 to
m cat
CM
_
VH] I.
N
ca
1i-o C
a
6
2
binding to the microtitre plate was blocked by incubation with casein (BDH, Poole, UK) (2'%, (w/v) in PBS). PBS containing 001(X Tween-20 was used for all washing procedures. Bound C4l I I I human antibody was revealed by incubation with alkalinephosphatase conjugated goat anti-human IgG or IgM (Sigma) (diluted 1:1000 in PBS containing 0 01% Tween-20) and detected by incubation in p-nitrophenol substrate (Sigma). 5 @ . In some instances there were modifications to this technique. Anti-collagen and anti-cardiolipin ELISA were carried out at room temperature. Cardiolipin was coated to microtitre plates by dissolving in ethanol and air-drying onto the plate. Tween-20 -_.was not used in the washing or dilution buffers for anti' ELISA. All ELISA results were compared with the binding of the .. .... _. . .- ... _supernatants to microtitre plates that had been incubated with casein, but not coated with antigen. Indirect immunofluorescent (IIF) staining techniques were used to measure the reactivity of undiluted culture SN with nuclear and cytoskeletal components of HEp-2 cells and 3T3 fibroblasts respectively and with double-strand DNA in Crithi_- ...... '- _ dia lucillae on multitest slides (Biodiagnostics, Malvern, UK). Standard IIF staining techniques were used with 20-min incubations at room temperature. A FITC-conjugated goat anti-human immunoglobulin (diluted 1:50 in PBS) was used to reveal human antibody bound to the substrate. IgM-RF and IgG-RF in culture SN has been measured previously [6]. t
0 4 4: 4 I l II I I
*
s . ~~~~~~~~~~~~cardiolipin
VH2
VH3
VN 4
*
Vat 5 t
SRESULTS
VHG6
Fig. 1. Total RNA extracted from each hybridoma was probed sequentially with each of the DNA probes specific for the human VH gene families 1-6 and for IgM and IgG C regions (Cp and Cy). Hybridization with the C region probes confirmed the presence of immunoglobulin messenger RNA. The VH probes did not crosshybridize when the filters were washed at high stringency. A representative panel of 10 hybridomas is shown here. UK) at 10 pg/ml in PBS by incubation at 40C overnight. All subsequent incubations were for 1 h at 370C. Non-specific
VH gene family usage in synovial-derived B cell hybridomas
Hybridizing bands of 2-4 and 2-2 kb with C region probes (p and y respectively) ascertained the presence of immunoglobulin message in the total RNA extracted from each hybridoma. RNA from each hybridoma hybridized with only one of the six VH gene probes (Fig. 1). The relative expression of each VH gene family in IgM- and IgG-secreting synovial-derived hybridomas is depicted in Table I . VH genes from families VH 1, VH2, VH3 and VH4 were represented in the 18 hybridomas, VH genes from VHS and VH6 families were not expressed.
Table 1. VH gene usage in IgM-, IgG- and IgA- secreting hybridomas
Hybridomas derived from non-rheumatoid spleen and tonsil
Hybridomas derived from rheumatoid synovium VH gene
family I 2
3 4 5 6
IgM-secreting (n=8) 1 (1250%) 1 (125-%) 3 (37.50/) 3 0 0
(37*5%")
IgG-secreting (n= 10)
IgM-secreting (n=7)
IgG-secreting (n= 11)
IgA-secreting (n=3)
2 (20%,)
1 (143'%)
3 (273%/o)
0
0
0 5 (71
0 5 (45
0 1
4 (40%Y) 4 0 0
(40%.)
4'%)
0 1(143%.) 0
50%)
2 (18 2°%) 1(91-%) 0
(33 3%) 2 (66-7%)
0 0
All hybridomas were derived by fusing human mononuclear cells with a heteromyeloma cell line (SPAZ-4). VH gene family expression was determined by Northern blotting using DNA probes specific for each of the six human VH gene families.
Immunoglobulin VH gene utilization in RA synovium
233
Table 2. The frequency of VH gene expression in a panel of hybridomas derived from rheumatoid synovial tissue and non-rheumatoid spleen and tonsil, compared with the frequency of VH family expression observed in the peripheral blood of normal individuals
VH gene expression Hybridomas
Peripheral blood
VH gene family
Size in the genome (predicted frequency of expression, %)
Family
Rheumatoid synovial-derived hybridomas (n = 18)
hybridomas (n=21) 4 (19%) 0 11 (52 5%) 4 (19%) 2 (9-5%Yo)
3 (16-7%) 1 (56%) 7 (38-9%) 7 (38-9%)
20-25 (33) 5-10(11) 25-30 (40) 6-10 (11) 2-3 (4) 1 (1)
1 2 3 4 5 6
Non-rheumatoid spleen- and tonsil-derived
0 0
0
PBMC
EBV-lines
EBV-lines
By in situ hybridization [7]
by Northern blotting [8]
11% 3%
12% 0%
52% 19% 10% 5%
56%
15%
