* Division of Rheumatology and Clinical Immunology, Department of Medicine, Analytical Toxicology Core Laboratory, Department of Physiological Sciences, and
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, Florida 32610-0221
Received October 9, 2003; accepted December 19, 2003
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ABSTRACT |
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Key Words: mineral oil; pristane; autoimmunity; autoantibodies; antinuclear antibodies; lupus.
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INTRODUCTION |
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Pristane (2,6,10,14-tetramethylpentadecane) and mineral oil induce plasmacytomas in susceptible strains of mice (Anderson and Potter, 1969). Pristane, incomplete Freund's adjuvant (IFA), and squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene) induce chronic arthritis in mice and rats (Cannon et al., 1993
; Carlson et al., 2000
; Wooley et al., 1989
). We have recently reported that, in addition to pristane (Satoh et al., 1995
; Satoh and Reeves, 1994
), IFA and squalene, but not medicinal mineral oils, can induce lupus-related anti-nRNP/Sm and Su autoantibodies in non-autoimmune-prone strains of mice (Satoh et al., 2003a
). These data suggest that hydrocarbons can have a variety of immune effects.
In the present study, we have analyzed various types of hydrocarbon by gas chromatography/mass spectrometry (GC/MS) and contrasted the immune response caused by medicinal oils and adjuvant oils. All hydrocarbons, including medicinal oils, induced hypergammaglobulinemia as well as autoantibodies, but the pattern was different. Since humans are exposed to a variety of hydrocarbons in daily life, there may be implications for the pathogenesis of autoimmune disease.
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MATERIALS AND METHODS |
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Analysis of mineral oils by gas chromatography/mass spectrometry (GC/MS).
A 100-microliter sample was diluted to 10 ml in hexane for analysis using an HP6890 gas chromatograph coupled to an HP5973 mass selective detector (Hewlett Packard Company; Wilmington, DE). The components of the injected sample (1 µl) were separated across an HP-5MS column (30 m x 0.25 mm; 0.250 mm film thickness) under an oven program that ramped from an initial temperature of 60°C at 10°C/min to 270°C (5 min hold); then increased at 5°C/min to 300°C (20 min hold). Each analyte of interest (pristane, n-hexadecane, and squalene) was quantified against a five-point standard curve (R2 0.9999). Quantification was based on the abundance of the analyte target ion (pristane and n-hexadecane, m/z = 57; squalene, m/z = 341) in ratio with the internal standard pyrene (m/z = 202).
Immunoglobulin ELISA.
Serum immunoglobulin levels at 3 months were measured by sandwich ELISA as described (Hamilton et al., 1998). Levels of IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM were determined using 1:200,000 and/or 1:500,000 sera using mouse immunoglobulin isotype controls as standard (mouse immunoglobulin panel, Southern Biotechnology, Birmingham, AL).
Anti-ssDNA antibody and anti-chromatin antibody ELISA.
Levels of anti-ssDNA and chromatin antibodies in sera (1:250 dilution) from BALB/cJ mice 3 and 6 months after treatment were tested by ELISA using calf thymus ssDNA (Sigma) and chicken chromatin, respectively, as antigen (Satoh et al., 2000b). Chicken chromatin was purified from chicken erythrocytes (Pel-Freeze Biologicals, Rogers, AR) as previously described (Sung et al., 1977
). Positives were defined as samples showing OD higher than the mean + 3 SD of 10 blank wells.
Immunofluorescent antinuclear antibodies (ANA).
The levels of antinuclear antibodies in sera from BALB/cJ mice at 3 and 6 months after treatment were determined by indirect immunofluorescence using L929 (mouse fibroblast) cells as described (Satoh et al., 1996). Sera were screened at a 1:40 dilution, and the titers were estimated using a titration emulation system ImageTiter (RhiGene, Inc., Des Plaines, IL) (Yoshida et al., 2002
).
Statistical analysis.
Frequencies of autoantibodies were compared by Fisher's exact test. Levels of immunoglobulins and autoantibodies were compared by the Mann-Whitney test.
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RESULTS |
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DISCUSSION |
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Intraperitoneal injection of pristane induces plasmacytomas (Anderson and Potter, 1969), destructive arthritis (Wooley et al., 1989
), and a lupus-like autoimmune syndrome (Satoh et al., 1995
; Satoh and Reeves, 1994
) in nonautoimmune strains of mice. IFA and squalene also induce autoimmune chronic arthritis in mice and rats (Cannon et al., 1993
; Carlson et al., 2000
). In addition, we recently reported that intraperitoneal injection of IFA or the adjuvant oil squalene (MF59) (O'Hagan et al., 1997
), but not medicinal mineral oils, can induce lupus-related autoantibodies to nRNP/Sm and Su, like pristane (Satoh et al., 2003a
). GC/MS analysis of hydrocarbon oils revealed that IFA contains 10- to 20-fold more pristane and n-hexadecane than medicinal oils (Table 2), though this represents only ~0.1% (w/v) of IFA. Considering the fact that various hydrocarbons, including pristane, squalene (Satoh et al., 2003a
), and n-hexadecane (Y. Kuroda et al., manuscript in preparation), can induce lupus autoantibodies, it is possible that induction of these autoantibodies is not due to the presence of traces of pristane in IFA. Instead, the present data suggest that the induction of autoantibodies may correlate better with the amount of C15C25 hydrocarbons present in an oil. Consistent with this interpretation, the medicinal oil that most efficiently induced autoantibodies was MO-F, the one with the highest level of C15C25 hydrocarbons. Interestingly, hydrocarbons with these carbon numbers (C15C25) represent the optimal size for adjuvanticity, as well (Whitehouse et al., 1974
). Thus, pristane and n-hexadecane may be representative of a much larger class of hydrocarbons within the C15C25 range with the capacity to induce antinuclear or anti-cytoplasmic antibodies. However, it is clear that other factors are involved as well, since squalene (C30) also induces autoantibodies to nRNP/Sm and Su.
The pathogenesis of lupus in human and mice is believed to result from the interaction of genetic and environmental factors (Hess, 2002). Different subsets of lupus are associated with unique symptoms with different autoantibodies (Reeves and Satoh, 2001
). However, the mechanisms remain poorly understood. It has been shown that low-affinity polyreactive IgM autoantibodies produced by B-1 cells are regulated differently than high-affinity IgG autoantibodies produced by conventional B-cells (Reap et al., 1993
). Data from our laboratory strongly suggest that different pathways exist for different type of autoantibodies. IL-6 plays an essential role for anti-chromatin and -dsDNA antibodies (Richards et al., 1998
), whereas IFN is critical for anti-nRNP/Sm and antiSu (Richards et al., 2001
). In NZB/W F1 and CBA/N (xid) mice, anti-RNA helicase A (RHA) antibodies are produced spontaneously along with IL-4 and IL-6, but pristane treatment shifts the cytokine balance toward IFN and IL-12, suppresses anti-RHA production, and induces anti-nRNP/Sm and Su antibodies (Satoh et al., 2003b
; Yoshida et al., 2002
). The lpr gene induces high levels of anti-chromatin/DNA antibodies but not anti-nRNP/Sm or Su in C57BL/6 mice, whereas pristane treatment induces the latter antibodies with only low levels of the anti-chromatin/DNA antibodies (Satoh et al., 2000a
).
The present data indicate that, whereas some hydrocarbons (squalene, IFA) predominantly induce anti-nRNP/Sm and Su antibodies, other hydrocarbons (MO-F, MO-S) efficiently induce anti-chromatin/DNA antibodies but not anti-nRNP/Sm or Su. All hydrocarbons induced hypergammaglobulinemia, but the T-cell-dependent subclasses IgG1 and IgG2a were characteristic of pristane, IFA, and squalene, which induce anti-nRNP/Sm or Su autoantibodies (Satoh et al., 2003a). The T-independent subclasses IgG3 and IgM were prominent in medicinal oil-treated mice. The predominant increase in IgG3 and IgM along with possible enhancement of spontaneous anti-ssDNA/chromatin antibody production was consistent with what is seen in silicone oil-treated mice (Naim et al., 2000
). The former oils are associated with early IL-12 production, whereas the latter group also induces IL-6 and TNF-
, especially at late stages of inflammation (Satoh et al., 2003a
). These data suggest that different mechanisms drive the production and regulation of subsets of autoantibodies induced by different type of oils. It is possible that high molecular weight hydrocarbons (>C25) are prone to induce anti-chromatin/DNA antibodies, whereas lower molecular weight hydrocarbons (C15C25) with adjuvant activity efficiently induce autoantibodies to nonchromatin antigens such as nRNP/Sm and Su, perhaps through the more efficient recruitment of T-cell help, reflected in the shift from IgM/IgG3 to IgG2a or IgG1. We suggest that the production of autoantibodies in this model is not simply the consequence of nonspecific inflammation, but rather it involves mechanisms specific for particular antigens, which maybe differently recognized in mice treated with different hydrocarbons.
The question arises of the significance of these findings for the pathogenesis of human disease. It might be argued that humans are not exposed to hydrocarbons via the intraperitoneal route and that other routes of exposure pose less risk. For instance, the immunotoxicity of aminocarb varies depending on the route of administration, with intraperitoneal exposure resulting in greater toxicity than oral administration (Bernier et al., 1995). Humans are exposed to mineral hydrocarbons via ingestion (foods, medications) (Dincsoy et al., 1982
; Grob et al., 1997
; Heimbach et al., 2002
), inhalation (diesel exhaust, oil mists, aspiration of ingested mineral oil) (Simpson et al., 2003
; Spickard and Hirschmann, 1994
), skin absorption (cosmetics, skin contact with oils or fuels) (Nash et al., 1996
; Riviere et al., 1999
), or injection (immunization, accidental inoculation) (Di Benedetto et al., 2002
; O'Hagan et al., 1997
). It is not known with certainty whether nonperitoneal exposure to hydrocarbons can induce autoimmunity. However, it is clear that such exposure can cause an intense inflammatory reaction (lipogranulomas) in the lungs, liver, and regional lymph nodes (Dincsoy et al., 1982
). Moreover, ingested mineral oil is absorbed through the intestine and becomes widely distributed throughout the body (Bollinger, 1970
; Ebert et al., 1966
). Mouse experiments are in progress to determine whether nonperitoneal exposure to pristane can induce the autoantibodies seen following intraperitoneal injection.
Another issue is whether the hydrocarbon doses given to the mice are ever reached in humans. Although the single 0.5-ml ip dose given to mice is quite large, humans can be exposed to considerable quantities of hydrocarbon oils over the course of a lifetime. It has been estimated that an average person living in a developed country ingests 50 grams of mineral oil per year in food (Heimbach et al., 2002). Individuals using mineral oil chronically as a laxative have much greater exposure. At the recommended dose of 13 tablespoons (1545 ml) per day, exposure may be as high as 0.75 ml/kg/day for a 60-kg person, or the equivalent of 20 µl/day in a mouse weighing 26.7 grams. On a kg-per-kg basis, the intraperitoneal dose given to the mice in these studies is comparable to that of a human ingesting mineral oil as a laxative for 25 days.
In summary, the present data indicate that the lupus-inducing hydrocarbons pristane, squalene, and n-hexadecane were present only in trace amounts in IFA or medicinal mineral oils. Although medicinal mineral oils did not induce anti-nRNP/Sm, or Su autoantibodies (Satoh et al., 2003a), they promoted anti-chromatin/DNA autoantibody production even more efficiently than squalene or IFA, suggesting that different types of autoantibodies could be generated in response to different hydrocarbons. Since mineral oils are nearly ubiquitous in the environment, whether these substances taken orally have a potential to trigger autoimmunity in human warrants further study.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Division of Rheumatology and Clinical Immunology, University of Florida, ARB-R2156, 1600 SW Archer Rd. Box 100221, Gainesville, FL 326100221. Fax: (352) 392-8483. E-mail: satohm{at}medicine.ufl.edu
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