Autoreactive responses to an environmental factor: 1. phthalate induces antibodies exhibiting anti-DNA specificity

Abstract

Environmental factors have been implicated in the induction of autoimmune disorders. We report here that a common chemical, phthalate, used widely in synthetic polymers and cosmetics induces serum anti-self DNA antibodies in BALB/c, NZB and autoimmune-prone NZB/W F1 mice. The latter group experiences a high mortality, and significantly higher anti-DNA antibody levels along with nephritis and other histopathologic changes in kidney. Comparison of amino acid sequences of an anti-phthalate BALB/c B-cell hybrid, 2C3 with the known database at the National Center for Biotechnology Information reveals a striking homology between the variable regions of 2C3-Ig (gamma1, kappa) and an anti-DNA antibody, BV04-01 (gamma2b,kappa) isolated from the lupus-prone NZB/W F1 mice. The homology is 98% for kappa light chain and 70% for gamma heavy chain. Like 2C3-Ig, BV04-01 also has specificity for d(pT)4. Furthermore, the light chains of both 2C3-Ig and BV04-01 are products of Vkappa1 gene. To understand the nature of anti-phthalate responses in general, hybridomas generated from phthalate-keyhole limpet haemocyanin-primed BALB/c splenocytes were characterized. The study identifies cross-reactive populations that strongly bind phthalate, DNA, or both. Of the 14 hybridomas evaluated, six express the same Vkappa1 gene-derived light chain as 2C3, and bind both phthalate and ds and ss-DNA. They specifically recognize the oligonucleotides, d(pT)4, and d(pT)10. Additionally, when antisera raised against idiopeptides corresponding to 2C3-Ig hypervariable regions are allowed to react with 2C3-Ig, their binding is blocked specifically by both d(pT)4 and phthalate. This study clearly demonstrates that phthalate exposure leads to activation of a significant number of autoreactive B-cells, with the consequence of a significant pathogenic progression in susceptible NZB/W F1 mice but not in non-autoimmune-prone BALB/c.

Figure 1

Comparison of amino acid sequences of the variable regions of light and heavy chains of 2C3 and BV04-01 antibodies. Dots indicate identity of amino acid sequences between BV04-01 with 2C3 and dashes denote regions lacking corresponding amino acid residues. CDR regions are underlined. A general numbering scheme introduced for immunoglobulins by Kabat et al. is used.

Figure 2

Inhibition of 2C3-Ig binding to ds-DNA was assessed in the presence of varying concentrations of homopolymers, d(pT)10, d(pA)10, d(pG)10, d(pC)10 and random hexamer primer (ss-DNA). Significant inhibition was raised with d(pT)10.

Figure 3

2C3-Ig bound not only to phthalate but also to d(pT)4. (a) Direct binding of 2C3-Ig to either phthalate-coated ELISA plate or d(pT)4-coated ELISA plate showed that 2C3-Ig had strong affinity to both phthalate and d(pT)4. (b) Inhibition of 2C3-Ig binding to d(pT)4 by phthalate. ELISA plate was coated with the oligonucleotide, d(pT)4, as described in Materials and Methods. 2C3 Ig-HRP conjugate was added in the absence of inhibitor (•), in the presence of intact 2C3-Ig (□), or phthalate (▴), as inhibitors. (c) The ELISA plate was coated with phthalate instead of d(pT)4, and tested as the same way as in (b).

Figure 4

Binding of 2C3-Ig to anti-idiopeptide antibody was inhibited by ligands. ELISA plates were coated with each idiopeptide-specific serum antibody (1 : 200 dilution), to which: (a) 2C3-Ig–HRP conjugate was added along with various concentrations of phthalate as inhibitor. (b) 2C3-Ig–HRP conjugate was added along with various concentrations of d(pT)4 as inhibitor (1 nm d(pT)4 = 1·5 µg).

Figure 5

Immunization with phthalate–KLH conjugates induces both anti-DNA and anti-phthalate antibody response in BALB/c, autoimmune-prone NZB/W F1, and parental NZB mice. The sera were tested at 1 : 200 dilution. The results represent average of two separate experiments (n = 8 mice in two experiments). (a) Anti-phthalate antibody levels in sera of mice immunized with phthalate–KLH were tested on phthalate–BSA-coated ELISA plates. (b) The anti-DNA antibody levels of the same groups. Sera obtained from BALB/c (adjuvant and KLH primed) and NZB/W F1 mice (unprimed and KLH primed) were used as control. *Significant decrease of anti-DNA antibody was observed in the serum of BALB/c mice at third immunization. (P < 0·05). 1. BALB/c immunized with phthalate-KLH conjugate. 2. NZB/W F1 immunized with phthalate-KLH conjugate. 3. NZB immunized with phthalate-KLH conjugate. 4. BALB/c immunized with adjuvant (CFA) only. 5. BALB/c immunized with KLH. 6. NZB/W F1 immunized with KLH.

Figure 6

Renal histopathology. Kidney tissues of three groups of mice at 7 month of age were examined following three weekly immunizations. (a) BALB/c mice immunized with phthalate–KLH emulsified in CFA; (b) NZB/W F1 mice immunized with CFA only; (c) NZB/W F1 mice immunized with phthalate-KLH emulsified in CFA. There were clear indications of increased leucocytic infiltration only in NZB/W F1 mice that were immunized with phthalate-KLH (c). haematoxylin–eosin stain; magnification, ×100.

Figure 7

Ligand (phthalate or DNA) binding with supernatants of hybridoma clones. White bars represent the binding with DNA; black bars represent that with phthalate. Antibodies after salt fractionation using 50% ammonium sulphate were used in ELISA at 50 µg/ml. All samples were assayed for binding to calf thymus DNA and phthalate. Results are average OD₄₉₀ (≥0·4) of two experiments, with 2C3-Ig, as positive control, and isotype-matched 1BF7 as negative control.

Figure 8

Cross-reactivity of monoclonal antiphthalate antibodies with DNA. (a) 2C3-Ig (γ1, κ) and L1B3-Ig (µ, κ) obtained from antiphthalate mAb producing hybridomas showed specificity to ds-DNA from calf thymus. (b) These two mAbs showed similar results to autologous cDNA from spleen of normal female BALB/C mice. 1BF7 and normal IgM antibodies were used as isotype-matched negative control. Results expressed as mean OD (490 nm) of triplicates ± SD are average of two separate experiments.

Figure 9

Vκ1 gene-specific mRNA expression in hybridoma clones. 13 hybridomas secreting either anti-DNA or anti-phthalate mAbs were screened for Vκ1 gene usage in their light chains. As described, the total RNA prepared from the hybridomas were reverse-transcribed using Vκ1-specific primers by RT–PCR and analysed by agarose gel electrophoresis. Lanes corresponding to each group (A to D) are as follows: A, 1: 100 bp marker, 2: Negative control minus reverse transcriptase, 3: 2C3, 4: L1B3. B, 1: 1A5, 2: 1G6, 3: 2G4, 4: 1H5. C, 1: 3D7, 2: 4A9, 3: 4C4, 4: 2G10. D, 1: 3B4, 2: 2D2, 3: 5C10, 4: 6D2.