Autoreactive responses to environmental factors: 3. Mouse strain-specific differences in induction and regulation of anti-DNA antibody responses due to phthalate-isomers

https://doi.org/10.1016/j.jaut.2005.04.002Get rights and content

Abstract

Little is known of the role of specific environmental factors in promoting autoimmune disorders such as systemic lupus erythematosus (SLE). This study addresses how exposure to phthalates, common environmental factors in foods, and biomedical devices could affect the immune functions of resistant and autoimmune-prone mice. We have previously shown that immunization with ortho-phthalate evokes anti-DNA antibody in BALB/c and NZB/W F1 mice, but only the latter suffer from nephritis and high mortality. BALB/c mice, in contrast, develop idiotype-specific CD8+ suppressor T cells downregulating autoreactive B cells. Here we report that all phthalate-isomers (ortho-, meta- and para-) are capable of inducing anti-DNA antibody responses and SLE-like syndromes. Kidney pathology worsens in NZB/W F1 and to a degree, in C57BL/6 mice after repeated exposure to phthalates. Only BALB/c and DBA/2 overcome adverse autoreactivity by induction of Ts cells; but in vivo depletion of these T cells renders these strains susceptible to autoreactivity. Anti-DNA antibodies in affected NZB/W F1 are largely IgG2a-type, while in BALB/c, DBA/2, and C57BL/6 mice IgG1-type. This is further corroborated by cytokine analyses that imply corresponding Th1/Th2 involvement. In summary, the commonly used phthalates appear harmful to susceptible strains, while BALB/c and DBA/2 are spared due to induction of Ts cells.

Introduction

The incidence of autoimmune disorder is on the rise, and in most cases, the etiology remains unknown. A case in point is systemic lupus erythromatosus (SLE), a disease characterized by high levels of self-reactive antibodies (Abs) directed to nuclear components including DNA, RNA and histones. While antibodies with these specificities may be generated in many individuals, only patients with SLE fail to regulate them effectively and consequently develop progressive renal disease. The exact molecular mechanisms underlying this pathophysiological process remain unknown. Factors, both inherent and environmental, have been implicated in the development of the disease [1], [2]. A number of studies suggest that foreign antigens exhibiting molecular mimicry of self components, play a critical role in triggering activation of autoreactive B and T cells [3], [4]. Activation of self-reactive clones of T or B cells may also occur during exposure to chemicals that are abundantly present in the environment, administered as medications or presented to the immune system [5], [6], [7].

Phthalates (i.e., the anion of phthalic acid) comprise a group of chemicals that are potentially autoantigenic like other haptenic molecules such as penicillin [8]. There is increasing evidence in both humans and rodents of exposure to phthalates that may cause adverse effects by initiating inflammatory responses and allergy-related diseases such as asthma and rhinitis [9], [10], [11], [12]. Phthalic acid and phthalate esters, such as 1,2-diethylhexyl phthalate (DEHP), are used in the production of a variety of household and consumer goods; including plastic polymers used in food-packaging materials, biomedical devices, children's toys, lubricating oils, and carriers for perfumes in cosmetics [13], [14]. Phthalate compounds are present in water, soil, food and even in the human body [15], [16]. ortho-Phthalic acid, the predominant metabolite of DEHP leaches out of polyvinyl chloride (PVC) plastics and appears in the urine of uremic patients receiving continuous ambulatory peritoneal dialysis [17], [18], [19], [20]. The biological consequences of these high levels of phthalate in blood and urine are currently unknown.

We have recently described molecular mimicry by ortho-phthalate of self-DNA and particularly, d(pT)4 nucleotides [21]. Antibodies induced by phthalate administration display cross-reactivity toward single and double-stranded DNA in both nonautoimmune BALB/c and lupus-prone NZB/W F1 mice [21]. These anti-phthalate antibodies also share idiotype identity with a well described anti-DNA mAb, BV-04 [22]. Interestingly, phthalate administration causes progressive renal failure and lupus-like affliction only in susceptible NZB/W F1 mice. These pathologies do not occur in resistant BALB/c mice due to induction of CD8+ suppressor T cells (Ts) that recognize the idiotype of phthalate-induced anti-DNA antibody light chain of predominantly Vk1 type. The Ts appear to be capable of deleting or suppressing autoreactive B cells before they could exceed the threshold by causing pathologic changes or a related malaise in BALB/c strains [23]. ortho-Phthalate, a DEHP metabolite, can thus be regarded as an autoantigen with ability to engender lupus-like autoimmune disorders.

Our findings with ortho-phthalate indicate that phthalates are good model system to study molecular mimicry of self components by environmental antigens and the factors that regulate susceptibility in various mouse strains. In the present study, we examined the relative autoantigenicities of all phthalate-isomers (ortho-, meta-, and para-). para-Phthalate is of particular interest because it is commonly used to make polyesters and plastic films. We report here that all phthalates are capable of inducing anti-DNA antibody responses. In BALB/c and DBA/2 mice, anti-DNA Ab levels declined following repeated phthalate immunization, and no lupus-like syndromes developed, possibly because of Ts induction. In contrast, C57BL/6 and NZB/W F1 mice develop higher levels of anti-DNA antibodies with the lupus-like syndromes and renal pathologies.

We also report that the pathogenic autoantibodies in susceptible strains belong to the IgG2a and IgG3 subclasses as has been reported by others [24], [25], [26]. Induction of these Ig subclasses is an indirect measure of the relative amount of Th1 and Th2 type cytokines. Several laboratories have demonstrated opposing roles of type 1 (IL-2 and IFN-γ) and type 2 (IL-4, -5 and -10) cytokines in expression of human and mouse models [27], [28]. Our report reveals a positive correlation between the cytokine profile observed and the subclasses of cross-reactive anti-DNA Ab induced.

Section snippets

Animals

Four strains of female mice, 8–12 weeks old, were used in this study: BALB/c, DBA/2, C57BL/6 and NZB/W F1. All strains of mice were obtained from Harlan Sprague Dawley (Indianapolis, IN), and maintained at the Indiana State University animal Facility according to principles of laboratory animal care (NIH publication 85-23) using protocols approved by the Indiana State University Animal Care and Use Committee.

Chemicals

Reagents and kits used in this study include the following: 4-aminophthalate (ortho

Assessment of toxicity of DEHP in autoreactivity and renal failure

To study the effects of long-term exposure to a commonly occurring phthalate ester DEHP, BALB/c mice were injected intraperitoneally with DEHP. Two groups of four mice were injected with 7.5 mg (0.5% of LD50) or 0.75 mg (0.05% of LD50), three times, respectively. As shown in Fig. 1., BALB/c mice injected with DEHP developed high levels of serum anti-DNA Abs in a dose-dependent manner. Moreover, 4–6 weeks after injection, the mice showed high levels of urinary protein and BUN (Table 1) as signs of

Discussion

The loss of tolerance to self-Ags that is characteristic of many autoimmune disorders remains poorly understood. Our study implicates phthalate-isomers, ubiquitous environmental factors, in initiation or exacerbation of SLE-like syndromes which are characterized by progressively deteriorating nephritis and anti-DNA humoral responses. This chronic disease strikes selectively, and genes certainly play important roles in susceptibility to this disorder [1], [2]. However, not all individuals who

Acknowledgements

The authors thank Professors William Brett, James Hughes, the graduate assistant Mr. Lavakumar Kalyampudi of the Department of Life Sciences and Mr. Thomas Rogge from the Office of Sponsored Programs at Indiana State University for their comments on the manuscript.

This work was supported by NIH R15CA86905, Grants from Indiana Academy of Sciences, and by Indiana State University Graduate student research fund (to S.-Y. L).

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