Technical Comments

Response to Comment on "The Influence of the Proinflammatory Cytokine, Osteopontin, on Autoimmune Demyelinating Disease"

Science  21 Mar 2003:
Vol. 299, Issue 5614, pp. 1845
DOI: 10.1126/science.1080223

Blom et al. (1) challenge several studies (2–6) that showed that osteopontin (OPN) plays a key role in the progression of autoimmune disease in models of multiple sclerosis (MS) and rheumatoid arthritis. According to Blom et al., the results of the study by Chabaset al. (2) and those reported in four other papers (3–6) reflect a polymorphism linked to OPN, rather than a role for the OPN protein itself. In this response to their comment, we review the results of the Chabas et al. (2) study; offer new data showing that vaccination against OPN potently modulates experimental autoimmune encephalomyelitis (EAE), a model of MS; suggest some possible reasons for the difference between the Blom et al. (1) results and our own (2); and note some additional studies that reveal that OPN is involved in the pathophysiology not only of MS but also of other neurological diseases (7–9).

Chabas et al. (2) reported that large-scale sequencing of non-normalized cDNA libraries derived from plaques dissected from autopsies of three brains of individuals with MS revealed that OPN transcripts were among the most abundant. OPN transcripts were likewise elevated in gene microarray analysis of EAE brain lesions. OPN was demonstrated in both MS and EAE lesions with immunohistochemistry. Within active MS plaques, OPN was found on microvascular endothelial cells and macrophages and in white matter adjacent to plaques. Reactive astrocytes and microglia also expressed OPN.

Progressive paralysis ensues in mice after injection of myelin oligodendroglial glycoprotein (MOG) peptide 35-55 in complete Freund's adjuvant (CFA). In OPN−/− knockout mice maintained on a 129/C57BL/6 outbred strain (10), injected with MOG 35-55, progressive EAE is rare, whereas remissions of disease are common. EAE was observed in 100% of both OPN+/+ and OPN−/− mice with MOG 35-55, but the severity of disease was reduced in all animals in the OPN−/− group, and these mice were totally protected from EAE-related death (2). During the first 26 days, OPN−/− mice displayed a distinct evolution of EAE, with a much higher percentage of mice having remissions compared to the controls.

Differences in cytokine expression in these mice confirmed that OPN was pivotal in controlling Th1/Th2 polarization. T cells in OPN−/− mice showed a reduced proliferative response to MOG 35-55 compared with the response of OPN+/+ T cells. In addition, IL-10 production was increased in T cells reactive to MOG 35-55 in OPN−/− mice that had developed EAE compared with T cells in OPN+/+ mice. At the same time, γ−IFN and IL-12 production were diminished in cultures of spleen cells stimulated with MOG (2). Futhermore, we recently showed that immunization with a DNA plasmid–encoding osteopontin (11) alters the chronic course of EAE (Fig. 1A). DNA immunization elicited antibodies to OPN, and disease modulation occurred at a time when antibody titers to OPN were highest (Fig. 1B).

Figure 1

(A) Immunization with DNA encoding OPN ameliorated MOG p35-55–induced EAE in C57BL/6 mice when administered before EAE induction. Mice were immunized weekly, intramuscularly for four weeks, with 100 μg of DNA vaccine encoding OPN, vaccine without DNA encoding OPN, or PBS only (16). Five days prior to first vaccination, mice were injected once with 10 μM cardiotoxin (Sigma). EAE was induced 1 week after the last vaccination by immunization with 100 μg of MOG p35–55 emulsified in CFA containing 4mg/ml heat-killed Mycobacterium tuberculosisH37Ra (Difco Laboratories, Detroit, MI). On the day of immunization and 48 hours later mice were injected with 100 ng of Bordetella pertussis toxin (BPT) in PBS, intravenously (i.v.). Mice were examined daily for clinical signs of EAE and scored as follows: 0, no paralysis; 1, loss of tail tone; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb and forelimb paralysis; and 5, moribund or dead. Each group included 10 female mice 4 to 5 weeks old. Asterisk (*) denotes p value <0.05, dagger (†) denotesp value <0.001, in comparing OPN DNA vaccine group with either of control groups. (B) Anti-OPN antibody production was detected in sera following OPN DNA vaccination. At different time points (0, 5, 14, 20, 30, and 40 days after EAE induction), sera were collected and anti-self-OPN antibodies were measured. OPN antibodies were detected after the DNA vaccination and boosted after EAE induction. The specific antibody titer was detected using a direct enzyme-linked immunosorbent assay (ELISA) (16). Results are shown as mean log2 of five different samples. Asterisk (*) denotes p value <0.002, dagger (†) denotesp value <0.0001, in comparing OPN DNA vaccine group with either of control groups.

Jansson et al. (5) described concordant results in another model of relapsing EAE—induced by proteolipid protein peptide (PLP) p172-183—in C57BL/6X129 OPN−/−knockout mice that had been backcrossed to C57BL/6 for six generations. Incidence of clinical disease and the mean day of onset were similar in OPN−/− and control, wild-type (OPN+/+) littermates. The OPN−/− animals, however, had lower mean maximal clinical scores and faster recovery without spontaneous relapses; moreover, they displayed decreased inflammatory infiltrates and demyelination, and their CD4+ T cells produced less γ−IFN and tumor necrosis factor–α. Taken together, these analyses of the effect of targeted mutations of OPN (2, 5) in two different EAE models indicate that OPN is likely to be a potent modulator of autoimmune demyelinating disease.

The divergent results described by Blom et al. (1) may be due to differences between the EAE model that they studied and the models used by our group (2) and by Jansson et al. (5). Blom et al. induced EAE with recombinant rat MOG 1-125, whereas we used a peptide fragment of MOG 35-55, which is the immunodominant epitope for B6. Lyons et al. (12, 13) showed that “the form of inducing antigen (protein versus peptide) plays a role in the pathogenesis of EAE,” (12) such that B cell–deficient mice did not develop EAE when immunized with MOG, although they were susceptible to MOG peptide 35-55–induced disease. Thus, different mechanisms are likely at work in EAE induced with recombinant proteins compared with the mechanisms in EAE induced with peptide fragments of the same protein. Furthermore, the recombinant MOG 1-125 of rat origin, used by Holmdahl and colleagues, has many differences compared with the mouse sequence.

Finally, to definitively implicate linked genes as the basis for differences between the data of Blom et al. (1) and those of our group (2) and Jansson et al. (5), EAE induced in earlier backcrosses of the Blomet al. OPN−/− strain would have to be examined. Until such differences are explicitly analyzed, it is difficult to draw any definitive conclusions. It is not at all unusual for divergent results to appear in studies of autoimmunity in knockout mice, especially when different animal models are examined (14).

Further recent studies, examining OPN polymorphisms and disease course in MS, support a role for osteopontin in MS. In 821 MS patients, a trend for association with disease course was detected in patients carrying at least one1284A allele in the OPN gene. Patients with this genotype were less likely to have a mild disease course and were at increased risk for a secondary-progressive clinical type (15). In addition, OPN transcripts were elevated in Huntington's disease, and transcripts and OPN protein were increased in the brain in a transgenic mouse model of Huntington's following successful treatment with cystamine (7). OPN transcripts and protein were also increased in an experimental model of epilepsy (8, 9).

In sum, the role of OPN in a wide variety of neuroinflammatory and neurodegenerative conditions remains an area worthy of intense activity. The conflicting results from Blom et al. (1) in the EAE model they used are of interest—but whether the differences are due to linked genes remains an open question at present.


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