Journal of Autoimmunity (1991) 4,665-679

Human Monoclonal Antibodies Demonstrate Polyreactivity for Histones and the Cytoskeleton

B. Joan Miller, John D. Pauls and Marvin J. Fritzler Department

of Medicine,

Faculty of Medicine, University Alberta, Canada

of Calgary,

Calgary,

(Received 16 November 1990 and accepted 18 March 1991) Systemic lupus erythematosus (SIX) and other autoimmune diseases are characterized by immune responses to intracellular, highly conserved antigens such as DNA and histone. In this study, peripheral blood lymphocytes (PBL) from a patient with histone autoantibodies were used to prepare IgM human-human hybridoma cell lines. Indirect immunofluorescence (HP) was used to identify monoclonal antibodies that bound to cytoskeletal and other cytoplasmic constituents. These supernatants did not bind doublestranded or single-stranded DNA. However, immunoblotting revealed that 7/20 hybridomas selected for their binding to cytoskeletal components produced antibodies that also bound mammalian and avian histones. When peptide fragments of histone were used in immunoblotting experiments, it was found that the monoclonal antibodies bound to the carboxyl terminus of Hl, a region previously shown to bind autoantibodies from sera of patients with SLE and drug-induced lupus (DIL). When the amino acid sequences of histones and cytoskeletal components were compared using the Swiss-Prot protein data bank, it was confirmed that there are eight regions of similarity. While the significance of polyreactive human monoclonal antibodies to cytoskeletal components and histones is not understood at present, it is possible that the human histone antibodies represent polyreactive antibodies that arise through the mechanism of molecular mimicry.

Introduction Systemic lupus erythematosus (SLE) and other autoimmune diseases are characterized by immune response to intracellular, highly conserved antigens such as DNA and histone [l-4]. Human monoclonal antibodies directed against nuclear antigens This research was supported by the Medical Research Council of Canada. Correspondence: Dr M. J. Fritzler, Department of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, AB T2N 4N1, Canada. 665 0896-8411/9

l/O40665 + 15 $03.00/O

0 1991 Academic Press Limited

666 B. JoanMiller et al. such as DNA, cardiolipin and other cellular antigens have been derived from many sources. These sources include stimulated and unstimulated tonsillar lymphoid cells and peripheral blood lymphocytes from normal individuals or patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [5-131. The study of human monoclonal antibodies to intracellular autoantigens has provided new insight into the autoimmune process. For example, the B cell repertoire has been more clearly elucidated because a wide range of monoclonal antibodies can be obtained from individuals with no record of exposure to the immunogen being studied, or a history of disease. In addition, these antibodies have been useful as probes of immunoglobulin gene usage and regulation [9]. Studies by Cote et al. [ 141 revealed that monoclonal antibodies to cytoplasmic and cytoskeletal structures outnumber those to membrane antigens. These observations emphasize the importance of intracellular antigens as the targets of autoimmune responses. Of relevance to this study, monoclonal DNA antibodies derived from SLE patients, as well as normal subjects, have been shown to bind to various cytoskeletal elements [ 15, 161. The cytoskeleton of most mammalian cells is composed of three main filamentous structures [17]. The 7-l 1 nm intermediate filaments have a size between that of the 22 nm microtubules and the 6 nm microfilaments. Intermediate filaments are further subdivided into five major types: cytokeratins, desmin fibres, glial filaments, neurofilaments and vimentin filaments. In this study, we have shown that monoclonal antibodies produced from lymphocytes of a patient with an undifferentiated connective tissue disease and circulating histone antibodies, primarily bound to cytoskeletal components, particularly intermediate filaments. Of importance, several of these same hybridomas produce IgM antibodies that demonstrated polyreactive binding to histones Hl, H3 and H4.

Materials and methods Production and screening of human-human monoclonal autoantibodies Fifty cc of blood was collected in heparin by standard venipuncture and the peripheral blood lymphocytes isolated on a Ficoll-Paque (Pharmacia LKB Biotechnology Inc.) gradient. The lymphocytes were then fused at a cell ratio of 1: 1 with the IgG,(k) producing GM4672 human lymphoblastoid cell line (Cell Repository Institute of Medical Research, Camden, N J, USA) using44.4% polyethylene glycol (Serva 1550) [6]. The fused cells were plated after 48 hours in 1 ml of a hypoxanthine, aminopterin andthymidine medium (HAT) in 2-ml wells at a cell concentration of 4 x 105/ml. The plated cells were left undisturbed for 7 days, at which time an additional 1 ml of HAT medium was added. Thereafter the HAT medium was replenished by one half the volume every 5 days. The supernatants were concentrated using an Amicon Centricon microconcentrator, and then applied to Low-Concentration Partigen Plates (Hoescht Canada Inc., Behring Diagnostics, Montreal, Canada) with appropriate standard controls. IIF and radial immunodiffusion confirmed that all hybridoma antibodies were immunoglobulins of the IgM isotype. Initial screening by indirect immunofluorescence (IIF) [18] employed freshly collected supernatants at 4 weeks post-fusion, a point in time when the hybridomas

Monoclonal antibodies bind cytoskeleton and histone

667

first became visible macroscopically. The conjugates used for IIF included polyvalent and monovalent fluorescein isothiocyanate (FITC)-conjugated antiserum, specific for human immunoglobulin IgG, IgA and IgM heavy and light chains, with an average fluorescein/protein (F/P) ratio of 2.8 (Hoescht Canada Inc.). HEp-2 cells (Immunoconcepts Inc., Sacramento, CA, USA) and U373 gliobastoma (American Type Culture Collection, Rockville, MD, USA) cells were used as substrates. Glioblastoma cells, chosen because they have been shown to express the intermediate filament antigens [19], were grown in RPM1 1640 medium supplemented with loo, fetal bovine serum, in a 5% CO, atmosphere at 37°C. Using IO-well, heavy, teflon microscope slides (Cell-Line Associates, Inc., Newfield, NJ, USA), glioblastoma cells were prepared for IIF substrate by culturing at 0.5 x lo6 cells in 30 ml medium per petri dish for 48 hours, rinsed with cold (4°C) potassium phosphate buffered saline (0.14 M NaCl, 0.01 M potassium phosphate, pH 7.2) and fixed in ice cold methanol ( - 20°C) for 5 min. The slides were then air-dried at room temperature prior to being used as substrates for IIF. Fluorescein isothiocyanate-conjugated mouse monoclonal antibodies to intermediate filaments (vimentin and cytokeratin), microtubule proteins and actin were used as controls to confirm cytoskeletal reactivity (Cytolite Kit, Biotechnology Systems, DuPont). A Crithidia Zuciliae immunofluorescence procedure was used to test the hybridoma supernatants for doublestranded DNA (dsDNA) antibodies [ 181. The intensity of IIF staining was graded as previously described [ 181. At 8 months, a total of 20 supernatants of monoclonal antibodies were selected on the basis of at least 3-4+ cytoskeletal binding positivity on IIF. One hybridoma with 2-3+ reactivity to large cytoplasmic granules was also included in the study as a control. Controls included an IgM monoclonal antibody directed against cardiolipin (gift of Dr J. Rauch, Montreal) and the supernatants of two non-secreting hybridomas from the same fusion plus supernatant from the fusion partner (human lymphoblastoid cell line, GM 4672). Sera from the PBL donor and from a patient with histone antibodies were included as positive polyclonal antisera controls. Electrophoresis,

transblotting

and immunoblotting procedures

Immunoblotting with different antigen sources was used as another method of identifying the reactivity of the monoclonal antibodies. Crude extracts were prepared from the glioblastoma U373 and HeLa cell lines by sonication of 5 x lo6 cells on ice in 1 ml of SDS-PAGE sample buffer (0.05 M Tris-HCI pH 6.8, 1 Osbsodium dodecyl sulfate, 191, 2-mercaptoethanol, 10% glycerol). Sonication included three 30 s pulses at 80 Hertz using a microtip, followed by centrifugation at 10,000 g for 30 min. The supernatant was collected and frozen at - 70°C until needed. Vimentin was isolated from the glioblastoma cell line U373 (ATCC), using the method of Starger and Goldman [20]. Commercially prepared human epidermal cytokeratin (Behring Diagnostics) and bovine skeletal muscle actin (Sigma, St Louis, MO, USA) were included in some immunoblots as reference controls. Chicken erythrocyte histones (Hl, H5, H2A, H2B, H3 and H4) were prepared according to published protocols [3]. Acetic acid hydrolysis of H 1 peptide fragments was performed using the modified methods of Neary et al. [21] and Sautiere et al. [22].

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Briefly, 2 mg of chicken erythrocyte Hl were dissolved in 0.2 ml of 0.25 M glacial acetic acid. The tube was evacuated, sealed and incubated at 105°C for 6 h. After breaking the seal, .the Hl digestion peptides were concentrated by vacuum centrifugation (Speed-Vat, Savant Instruments, Inc.). Trypsin-generated Hl peptide fragments were prepared by using a modified method from Aviles et al. [23]. Two mg of chicken erythrocyte H 1 were dissolved in 0.2 M K,SO,, 50 mu Tis-HCl buffer, pH 8. The optimal trypsin digest occurred at 3 min using 8 BAEE units of trypsin (Boehringer-Mannheim, Dorval, Que., Canada). The reaction was quenched using SDS-PAGE sample buffer. The proteins and peptides were separated by sodium dodecyl sulfate-18% polyacrylamide gel electrophoresis (SDS-PAGE) using a mini-gel apparatus (Bio-Rad Laboratories, Richmond, CA, USA). All immunoblot procedures, including the identification of histone peptide fragments, followed previously published methods [24]. An IgM peroxidase biotin avidin system (Vectastain ABC Kit, Vector Laboratories, Inc., Burlingame, CA, USA) was used and incubations with the undiluted human monoclonal antibodies were 3 h at room temperature followed by an overnight incubation at 4°C. Absorption studies Absorption of the hybridoma supernatants employed cytokeratin, melittin, histone and double-stranded and single-stranded DNA. Absorption was followed by standard IIF using a Hep 2 substrate. The ssDNA was prepared by boiling the DNA for 10 min in standard saline-citrate buffer (0.15 M NaCl, 0.015 M Na,C,H,O,) and cooling rapidly on ice [25]. Testing of the commercial calf thymus histone by SDSPAGE indicated that all the histones (Hl, H2a, H2b, H3 and H4) were present. A checkerboard series of varying dilutions of the antigen or the serum known to contain histone or DNA antibodies was also used as a control to monitor absorption. The supernatant of the hybridoma IF-13, diluted 1: 10 to 1:320, was absorbed overnight at 4°C with 75-100 ug/ml of cytokeratin (Calbiochem, San Diego, CA, USA) or 25-400 ug/ml of melittin (Sigma). The supernatant of hybridoma B-K6 was diluted l/20 to l/320 in PBS, pH 7.5 and absorbed with 30-500 l.tg/mlcalf thymus histones (Boehringer-Mannheim), dsDNA or ssDNA (Worthington Biochemical Corporation, Freehold, NJ, USA) for 1 h at 37°C then overnight at 4°C [15]. All samples were centrifuged at 14,000 g for 10 min prior to IIF analysis. The efficiency of histone antibody absorption was monitored by using the extraction-reconstitution technique [2]. Sections of cryopreserved mouse kidney, 4- to 6-u thick, were fixed in ice cold acetone for 1Omin at room temperature. The sections were then processed using the extraction-reconstitution protocol, followed by incubation with undiluted hybridoma supernatants using standard IIF procedures to identify histone antibodies. Protein sequence similarities IntelliGenetics (Mountain View, CA, USA) microcomputer software PC-Gene and the Swiss-Prot protein data bank were used to search for sequence similarities between histone components and other proteins.

Monoclonal antibodies bind wtoskeleton and histone

669

Table 1. Autoantibody reactivity of human hybridoma supernatants derived from lymphocytes of one patient as detected by indirect immunojluorescence

Post fusion (months)

Cytoskeletal components

Other components

Strongly positive cytoskeletal components (>3to4+)

3 8

45150 (90%) 40193 (43%)

5/50 (lo:,;)* 53/93 (57%)**

38/45 (84%) 24140 (60%)

*Others: 1 nuclear rim, 1 anti-large cytoplasmicspeckles,3 unidentifiable. **These were non-secreting or l-2+ positive speckled cytoplasm only, with no anti-cytoskeletalantibodies(4f being the maximum). Results

Hybridgrowth Fusions were plated at a density which allowed multiple colonies to grow in each well. At 4 weeks, macroscopic hybridoma clones were observed in 31 of 144 (21.5Yo) wells and of these, 12 of 144 (8.796) demonstrated weakly positive IIF reactions on HEp-2 substrate. At 5: weeks, 78 of 144 (540/,) wells were growing hybridomas macroscopically and 42 of these 78 (58.37;) supernatants were positive by IIF. Most of the hybridomas demonstrated weakly positive staining by IIF (1 to 2+) and 12 of 78 (15.4”;) wells contained hybridomas that produced strongly positive (3 to 4+) staining intensity. Hybridomas that demonstrated the strongest staining were cloned and subcloned by limiting dilution in liquid culture without feeder cells at one cell per well in a 96-well plate [26]. None of the hybridoma supernatants produced antibodies binding nuclear components as detected by IIF. Forty-five of the initial hybridoma supernatants demonstrated antibodies to cytoskeletal components, three hybridoma supernatants had IIF patterns consistent with actin or cytokeratin binding, one had a nuclear rim pattern, and one demonstrated a 4+ finely speckled cytoplasm. At 3 months, the majority (38/45 or 84Th) of the hybridomas producing cytoskeletal antibodies showed high titre and definite, highly resolved binding patterns on HEp-2 substrate (Table 1, Figure 1). Hybridomas selected for their cytoskeletal antibody binding on HEp-2 cells were also tested on the glioblastoma cell substrate. These also showed high titre staining of the cytoskeleton of the glioblastoma cells. As before, no nuclear staining was observed at this stage of cloning. Subsequent subcloning of the mixed and nuclear rim hybridomas demonstrated antibodies that reacted with the cytoskeleton (1 to 2 + IIF). One hybridoma (18B2), which initially demonstrated a coarsely speckled cytoplasmic staining pattern, later appeared to stain much larger cytoplasmic granules (LCG). None of the hybridoma supernatants had anti-dsDNA activity as measured by Crithidia lucillae assay. The hybridomas maintained in culture for 8 months or longer with subcloning continued to produce monoclonal antibodies that bound to intermediate filaments

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B. Joan Miller et al.

Figure 1. Indirect Immunofluorescence reactions (IIF) on HEp-2 cell substrate produced by donor sera, human monoclonal antibodies and controls. (a) Human donor sera (l/20 in PBS) demonstrates homogeneous and nucleolar staining patterns. (b) The undiluted supematant of the GM 4672 human lymphoblastoid fusion partner is negative by IIF. (c) Anti-histone control sera, diluted l/20, demonstrate a homogeneous nuclear staining. (d) Undiluted supematant of Hmab B-K6 demonstrating cytoskeletal staining. (e) Mouse anti-vimentin control. (f) Mouse anti-cytokeratin control ( x 250).

(24/40, 60% were strongly positive by IIF). The other hybridomas (53/93, 51%) became non-secreting or produced supernatants with markedly reduced staining (Table 1). With the exception of the LCG hybridoma 18B2 and three hybridomas that produced cytoskeletal antibodies, all of the antibody-producing hybridomas were of the IgM isotype as detected by radial immunodiffusion and IIF. The IgM concentrations of the 21 supernatants ranged from undetectable levels to 68.5 ug/m1/106 cells. The supernatant of the GM 4672, the fusion partner, the two non-secreting hybridoma controls, and the LCG hybridoma 18B2 did not produce a reaction in radial immunodiffusion. Immunoblotting The results of the immunoblotting assays are summarized in Table 2 and illustrated in Figure 2. Using an IgM secondary antibody, an immunoblot of HeLa cell extracts demonstrated the same 47 kDa band or multiple bands at the 47 kDa region on 14 of the 20 supernatants that demonstrated cytoskeletal binding by IIF. Of these 14, seven also reacted with Hl, H3 and H4. One reacted with Hl alone. The LCG hybridoma showed three weak bands of approximately 47 kDa, and a weak reaction

neg. neg. neg. neg. 31,15,11 kDa ND

neg. neg. neg. neg. 3 bands - 41 kDa, Hl, H3, H2a, H4 104 kDa, 3 bands 47 kDa, Hl, H3, H2b, H2a, H4

U373 Extract IB IgM neg. neg. neg. 47 kDa 58,31 kDa neg. 58,31,20 kDa 58 kDa 58,47,31 kDa 58,47 kDa 58,47 kDa 58,47 kDa 58,47,31 kDa 58,47 kDa neg. 58,31 kDa 58 kDa 58 kDa 58,31 kDa 58,31 kDa neg.

HeLa IB IgM

47

47 47 47 47 47

47 47

neg. neg. neg. neg. ND ND

neg. 47 kDa neg. kDa doublet kDa doublet neg. kDa doublet kDa doublet kDa doublet kDa doublet kDa doublet 47 kDa kDa doublet 47 kDa 47 kDa 47 kDa neg. neg. 47 kDa 47 kDa ND

Keratin IB IgM

reactivity of monoclonal antibodies and controls

neg. neg. neg. neg. 3 bands - 47 kDa, Hl, H3, H4 neg. 3 bands - 47 kDa, Hl, H3, H4 45,47 kDa, Hl, H3, H4 3 bands - 47 kDa, HI, H3, H4 45 kDa 47 kDa 47 kDa 3bands_47kDa,Hl,H3,H4 47 kDa 47 kDa 3 bands - 47 kDa, Hl, H3, H4 47 kDa 4 bands 65-80 kDa, Hl, H3, H4,24 kDa Hl, H3, H4 3 bands - 47 kDa, HI, H3 3 bands - 47 kDa, Hl, H3, H4

*IB = immunoblot, ND = not done.

A-IF8 A-IF13 A-IF18 A-IF22 A-IF24 A-K12 A-K13 A-K14 A-FI. 1 B-IF4 B-IF8 B-IF12 B-IF13 B-IF19 B-IF24 B-K6 B-K9 B-K1 1 B-K14 B-Fl. 1 18B2 Controls GM 4672 B-IF2 B-IF3 No antibody Donor serum Histone serum

Monoclonal antibody

Table 2. Immunoblot

neg. neg. neg. neg. HI, H5, H3, H2b, H2a, H4 Hl, H5, H3, H4

S Hl, H5 neg. Hl,H5 ND Hl, H5 ND ND ND Hl, H5 ND ND Hl, H5, H3, H4 ND ND Hl,H5 Hl, H5 neg.

neg. ND

Histone IB IgM

672 B. JoanMiller et al.

Western Blot of HeLa Whole Cell Extract stain

abed

gh

i

jklmnop

47 kdHlHK. H2ab H4-

Figure 2. IgM and IgG specific immunoblots against crude extract of HeLa cells demonstrating polyreactivity of the monoclonal antibodies with both histones and cytokeratins. Protein was applied to the gel at a concentration of 10 ug/lane. Stain: amido black stain oftransblotted crude extract of HeLa cells. Lanes a-d: control: includes no antibody (lane a), undiluted supernatants of fusion partner lymphoblastoid cell line GM 4672 (lane b), of non-secreting Hmab B-IF2 (lane c) and the IgM cardiolipin monoclonal antibody (lane d). IgG reactivity of the donor sera (lane e) and histone control (lane f) both diluted l/50 in Tris buffered saline (TBS), pH 7.4. IgM reactivity of the donor sera (lane g) and histone control (lane h) diluted l/50 in TBS. Supematants of human monoclonal antibodies demonstrating polyreactivity to multiple keratins in the 47kDa region and to histones, predominantly Hl, H3 and H4 (lanes i-o). Undiluted supematant of human monoclonal antibody that reacted with large cytoplasmic granules by IIF (lane p).

with H 1, H3 and H4 (Figure 2). When a secondary antibody of polyvalent immunoglobulins and commercial cytokeratin was used as the antigen, immunoblots of the undiluted supernatants from 15 of 20 supernatants that produced cytoskeletal staining by IIF demonstrated either a single or double band near the 47 kDa marker (data not shown). The seven supernatants that reacted with the histones in the crude HeLa extract were then tested by immunoblotting of the purified chicken erythrocyte Hl, H5 and core histones. Six of seven reacted with Hl and H5, and of these six, one also reacted weakly with Hl, H5, H3, and very weakly with H2b and H4. Immunoblots using a crude extract of U373 glioblastoma cells demonstrated a 58 kDa (14/20) and a 47 kDa band (7/20). Seven of the supernatants that bound to cytoskeletal proteins using the U373 cell extract also bound to Hl . The LCG hybridoma supernatant showed no reactivity in immunoblotting experiments. The sera of the lymphocyte donor (diluted l/50) immunoblotted against the HeLa extract showed three bands at 41 kDa as well as reactivity to Hl, H2a, H3, H4 and other proteins. The histone antibody control sera bound to all the histones and to other proteins including the 47 kDa protein (Figure 2, Table 2). The results of immunoblotting using Hl peptides are shown in Figure 3. Acetic acid reacts with peptide bonds in which the carbonyl group is contributed by aspartic

Western Blot of Trypsin Digested Hl Stain 1

e

abed

I

f

G-Hl-

Western Blot of Acetic Acid Hydrolysed Hl Stain 2

i

j

k

0

P

HAcH 11

Figure 3. IgM immunoblots of trypsin digested and acetic acid hydrolysed Hl. Trypsin digested Hl was applied to the gel at a protein concentration of 25 pg/lane, acetic acid peptides of Hl were applied at a protein concentration of 10 pg/lane. Stain 1: amid0 black staining of trypsin digested Hl which contains portions of globular domain and amino terminus (labelled G-HI) on a representative nitrocellulose strip after transfer. Lanes a-d: undiluted supematant of a non-secreting hybridoma (lane a), undiluted supernatam of the cell line GM 4672 (lane b), normal human sera diluted l/SO (lane c), and undiluted supernatant of the IgM cardiolipin monoclonal antibody. Lanes e-h: no binding to the globular domain and amino terminus by the fusion donor sera (lane e), the histone control sera (lane f) or the two human hybridoma supernatants B-K6 (lane g) and B-IF4 (lane h) respectively. Stain 2: amido black staining ofthe two peptides of acetic acid hydrolysed Hl which contain portions of globular and carboxyl terminus (labelled HAcHl). Lanes i-k: undiluted supematant of the cell line GM 4672 (lane i), normal human sera diluted 1/SO(lane j) and undiluted supematant of the IgM cardiolipinmonoclonal antibody (lane k). Lanes l-p: demonstrate binding to the acetic acid hydrolysed peptides of Hl by the control polyclonal human sera (lanes 1, m, n) and by the human hybridomas B-K6 (lane o) and B-IF4 (lane p).

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B. Joan Miller et al.

acid residues. With Hl, this is at amino acid 72, thus the two resulting peptide fragments include the carboxyl terminus and globular domain of the molecule. Trypsin digested Hl (amino acids 35-112) contains portions of the amino terminus plus the globular domain. The B-K6 and IF-4 supernatants, immunoblotted against the trypsin digested peptides of H 1, indicated no reaction with the amino terminus or globular domain. By contrast, the same supernatants reacted with two peptides derived from the globular domain and carboxyl terminus of Hl by acetic acid hydrolysis (HAcHl), thus indicating that the reaction was to the carboxyl terminus. None of the negative controls for the immunoblots stained the immunoblots at equivalent antibody concentrations. Absorption studies Absorption of the hybridoma supernatants with cytokeratin, melittin or histone decreased the intensity of the cytoskeletal IIF staining, whereas absorption with double stranded or single stranded DNA did not alter staining. The efficiency of histones as absorbents was based on the decrease of staining intensity on the histone reconstituted kidney substrates. Undiluted supernatants of hybridoma B-K6 shown to be positive by immunoblotting to cytokeratin, HI, H3 and H4 were first tested on untreated mouse kidney sections using an indirect immunofluorescence procedure. When the B-K6 supernatant was tested, the control treated slides demonstrated a 4+ cytoplasmic pattern with no nuclear staining. When histone and other soluble proteins were extracted with dilute acid, a decrease of cytoplasmic staining from 4+ to 2 + was demonstrated. The intensity of the cytoskeletal staining was not restored when the slides were reconstituted with histone. This suggests that histone antibodies may cross-react with cytoskeletal components and that histones are not bound to cytoskeletal components. As before, no nuclear staining was observed. The histone control sera was positive on the untreated substrates as expected, negative on the extracted section and positive on the reconstituted section as described previously [2]. Protein sequence similarities Since the immunoblotting and IIF data (Figures 1,2 and 3, Table 2) suggested that monoclonal antibodies that bound cytoskeletal components could cross-react with histones, the Swiss-Prot protein data bank was searched for sequence similarities between histone components and other proteins (particularly cytoskeletal proteins). (Table 3). Since not all of the cytoskeletal proteins have been sequenced, this analysis is only indicative of the potential for cross-reactivity. The analysis was performed with the PC-Gene program, using the Dayhoff MDM-78 matrix (‘k-tuple’ value of either 1 or 2 and ‘distance’ parameter of two amino acids). All histones showed a relative overall score of less than 8% identity with the intermediate filaments, with H4 having the greatest identity and Hl having the least identity. Evaluation of these regions of identity revealed a common tetrapeptide (GRPK) in the carboxy terminus of Hl (177-180) and the amino terminus of protozoa actin (35-38). A second common peptide (ATKKK) was identified in the carboxy terminus of H 1(118-l 22) and porcine neurofilament triplet L protein (543-547). Immunoblotting (Figure 3)

Monoclonal antibodies bind cytoslceleton and histone

675

Table 3. Sequence similarities between histones and cytoskeletalproteins Protein

Peptide

Sequence no,

Species

Hl Amino terminus actin Hl neurofilament triplet L H5 Cytokeratin H5 Cytokeratin H5 Cytokeratin H3 Actin H3 Tubulin H4 H5 Actin H4 Actin

GRPK GRPK

177-180 35-38

Chicken Protozoa

ATKKK ATKKK

118-122 543-547

Chicken Pig

RGGSS RGGSS SRGGS SRGGS DLQIKL DLQIKL EIRR EIRR IRKL IRKL IRRL IRRL IRRL RGVLK RGVLK

42-46 551-555 4145 150-154 65-70 149-154 50-53 325-328 62-65 122-65 34-37 72-75 326-329 55-59 61-65

Chicken Mouse Chicken Bovine Chicken Human Chicken Chicken Chicken Human, chicken, pig, rat Chicken Chicken Chicken Chicken Protozoa

also indicated that the cytoskeletal human monoclonal antibody reacted with the carboxy terminus of H 1. The source of histone for this study was chicken erythrocytes and thus it contained H5. It was interesting to note common peptides in both H5 and cytokeratins because immunoblotting (Table 2) also indicated reactivity with H5. A common pentapeptide (RGGSS) was found in the globular domain of H5 (42-46) and murine cytokeratin (551-555). A second common pentapeptide (SRGGS) was found in H5 (41-45) and bovine cytokeratin (15&154). A hexapeptide (DLQIKL) from the globular domain of H5 (65-70) was found to be similar to the hexapeptide (DLQAKL) of human cytokeratin (149-154). Of interest, other sequences of identity with H3 and H4 were found. An identical tetrapeptide (EIRR) was found in H3 (50-53) and avian actin (325-328). A second tetrapeptide (IRKL) was found in H3 (62-65) and human, avian, porcine and rodent tubulin (122-125). Analysis for other potentially cross-reacting determinants shows similarity to a tetrapeptide (IRRL) found in H4 (34-37) and H5 (72-75). The tetrapeptide (IRRL) from H4 (34-37) was also found in avian actin (326-329) and another H4 pentapeptide (RGVLK) (55-59) was found in protozoan actin (61-65).

Discussion

This study has described stable, human monoclonal cytoskeletal elements and histones. As demonstrated

antibodies that bind to by immunoblotting, the

676 B. JoanMiller et al. cytoskeletal binding supematants bound predominantly to intermediate filaments and to mammalian or avian histones. Although all the monoclonal antibodies studied bound to cytoskeletal or cytoplasmic components, no nuclear staining was detected by IIF. This is not an unusual finding since not all histone antibodies are detected by IIF on fixed substrates [24]. One explanation for this phenomenon is that some histone epitopes may be altered or not available for binding in fixed tissue culture cells. Alternatively, the purification of histones may expose determinants that are not available for antibody binding in situ. Several investigators have shown that monoclonal antibodies derived from patients with autoimmune diseases, as well as from healthy humans, demonstrate reactivity with cytoskeletal proteins [5-131, and polyreactivity with intermediate filaments and other macromolecules have been noted by others [27,28]. For example, monoclonal DNA antibodies have been shown to bind cytoskeletal proteins [ 151. This is of interest because a theme of autoantibody reactivity in systemic rheumatic diseases suggests that certain autoantibody pairs occur in common association or, what has been referred to as ‘linked sets’ [29]. This ‘linkage’ is expressed as reactivity against nucleoprotein macromolecules or structures. For example, patients with antibodies directed against DNA commonly carry antibodies directed against histones. This observation, along with studies of the determinants and epitopes bound by these sera, has suggested that the nucleosome is the immunogen in these patients [3, 301. Therefore, the observation that monoclonal antibodies directed against DNA and histone, the essential components of the nucleosome, also bind cytoskeletal elements, provides new insight into the nature of the (auto)antigens that may trigger and sustain certain autoantibody responses. One might dismiss these cross-reactivities as ‘trivial’ or insignificant, except to note that the determinants bound by the monoclonal antibodies BK-6 and B-IF4 are identical to those bound by the antibodies from patients with SLE and DIL [3,4]. In addition to polyreactivity of DNA antibodies, cross-reactions of histone monoclonal antibodies with lysosomal protease cathepsin B, soluble retinal protein, bacteriophage lambda structural head protein D, and to contractile proteins such as actin, myosin and tropomyosin have been described [31,32,33]. To our knowledge, there have been no other reports of monoclonal histone antibodies that bind cytoskeletal components. As in this study, the other polyreactive monoclonal antibodies were predominantly of the IgM antibody class [5, 14, 34, 351. Although the polyreactive binding of IgM monoclonal antibodies is a well established feature, it should not detract from the observation that the binding is relatively specific. As noted above, demonstration that the IgM antibodies in the study bound to the same Hl peptide fragments as human autoantibodies supports the importance of these findings. The human monoclonal histone antibodies in our study react with several forms of cytokeratin having molecular weights of approximately 47 kDa. The reactivity of some supernatants that bind Hl and vimentin were detected using a crude glioblastoma cell extract. Nineteen cytokeratins with molecular weights in the 4068 kDa range have been described [ 191. Other investigators have shown that vimentin has a molecular weight of 57 kDa, which is approximately that of the immunoblot band observed in our U373 extracts. Interestingly, Cote et al. [ 141 found that human monoclonal antibodies bound to proteins of 55 and 46 kDa. Therefore, the 47 and

Monoclonal

antibodies bind cytoskeletonaad histone 677

58 kDa reactivity of our monoclonals are probably directed against keratin and vimentin, respectively. All classes of intermediate filaments appear to share a common antigenic determinant [36,37]. Since the reactivity of human monoclonal antibodies is not restricted to intermediate filaments and tends to encompass all three major filamentous components, we cannot exclude binding to other cytoskeletal proteins [27]. The polyreactivity of the monoclonal antibodies that bind intermediate filaments and the histones suggest that these proteins share common epitopes. The similarities in the protein sequences between the histones and intermediate filaments, in particular cytokeratins, might be expected, when one considers that an antibody is capable of recognizing antigenic determinants comprised of four to six amino acids. Given that there are only 20 common amino acids and a large number of proteins present in man, it is possible that a similar sequence could appear by chance in two unrelated molecules [38,39]. Studies by Haspel et al. [40] suggested that monoclonal multiple organ reactive autoantibodies react with either the same protein present in several organs or with common determinants on different proteins in multiple organs, or that they may accommodate structurally similar but non-identical epitopes on different proteins. Garzelli [41] suggested that lymphocytes capable of making multiple organ reactive antibodies are a common feature of the normal B-cell repertoire of the host. Because the positively charged protein melittin, which bears no sequence similarity with histones, absorbed the human monoclonal antibody, some binding may occur as a result of the charge on the proteins. Lastly, current technology does not allow the investigator easily to assess conformational determinants on proteins. The observation that monoclonal cytoskeletal antibodies bind to histone might be owing to such determinants. While the significance of polyreactive human monoclonal antibodies to cytoskeletal components and histones is not understood at present, we suggest, as others have in the past, that the monoclonal antibodies may be detecting conserved recognition sites underlying the specific macromolecular interactions that control important biological processes [42]. Owing to the clinical significance of histone antibodies in SLE and other autoimmune diseases, studies of human monoclonal antibodies that react with histones may lead to further insights into the autoimmune process. Acknowledgements J. D. I’. is a recipient of a Canadian Arthritis Society Studentship and M. J. F. is a Scholar of the Alberta Heritage Foundation for Medical Research. This research was supported by the Medical Research Council of Canada. References Tan, E. M., E. K. L. Chan, K. F. Sullivan, and R. L. Rubin. 1988. Antinuclear antibodies (ANAs). Diagnostically specific immune markers and clues toward the understanding of systemic autoimmunity. Clin. Immunol. Immunopathol. 47: 121-141 Fritzler, M. J. and E. M. Tan. 1978. Antibodies to histones in drug-induced and idiopathic lupus erythematosus.3. Clin. Invest. 62: 560-567 Gohill, J., P. D. Cary, M. Couppez, and M. J. Fritzler. 1985. Antibodies from patients with drug-induced and idiopathic lupus erythematosus react with epitopes restricted to the amino and carboxyl termini of histone. 3. Zmmunol. 135: 3116-3 12 1

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