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Induction of arthritis by high mobility group box chromosomal protein 1 is independent of tumour necrosis factor signalling.

Pullerits R, Jonsson IM, Kollias G, Tarkowski A - Arthritis Res. Ther. (2008)

Bottom Line: HMGB1 is actively released from immune cells in response to TNFalpha; once released, HMGB1 in turn induces production of several proinflammatory cytokines--including IL-6 and TNFalpha--by macrophages.The purpose of the present study was to investigate whether the arthritis-inducing effect of HMGB1 is dependent on TNFalpha expression in vivo and to assess whether TNFalpha deficiency affects a proinflammatory cytokine response to HMGB1 in vitro.Our data show that HMGB1-triggered joint inflammation is not mediated via the TNF pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Rheumatology and Inflammation Research, Sahlgrenska Academy at Göteborg University, Guldhedsgatan 10A, 41346, Göteborg, Sweden. rille.pullerits@rheuma.gu.se

ABSTRACT

Introduction: TNFalpha and high mobility group box chromosomal protein 1 (HMGB1) are two potent proinflammatory cytokines implicated as important mediators of arthritis. Increased levels of these cytokines are found in the joints of rheumatoid arthritis patients, and the cytokines trigger arthritis when applied into the joints of naïve mice. HMGB1 is actively released from immune cells in response to TNFalpha; once released, HMGB1 in turn induces production of several proinflammatory cytokines--including IL-6 and TNFalpha--by macrophages. Whether HMGB1-induced arthritis is mediated via the TNFalpha pathway, however, is unknown. The purpose of the present study was to investigate whether the arthritis-inducing effect of HMGB1 is dependent on TNFalpha expression in vivo and to assess whether TNFalpha deficiency affects a proinflammatory cytokine response to HMGB1 in vitro.

Methods: TNFalpha knockout mice and backcrossed control animals on a C57Bl6 background were injected intraarticularly with 5 microg HMGB1. Joints were dissected 3 days after intraarticular injection and were evaluated histologically by scoring the frequency and severity of arthritis. For in vitro studies, mouse spleen cultures from TNFalpha knockout mice and from control mice were incubated with different doses of HMGB1, and cell culture supernatants were collected at different time points for analysis of IL-6.

Results: Intraarticular injection of HMGB1 into healthy mouse joints resulted in an overall frequency of 32% to 39% arthritic animals. No significant differences were found with respect to the severity and incidence of synovitis between mice deficient for TNFalpha (seven out of 18 mice with arthritis) in comparison with control TNFalpha+/+ animals (six out of 19). No significant differences were detected between spleen cells from TNFalpha+/+ mice versus TNFalpha-/- mice regarding IL-6 production upon stimulation with highly purified HMGB1 after 24 hours and 48 hours. Upon stimulation with a suboptimal dose of recombinant HMGB1, however, the splenocytes from TNFalpha+/+ animals released significantly more IL-6 than cells from the knockout mice (602 +/- 112 pg/ml and 304 +/- 50 pg/ml, respectively; P < 0.05).

Conclusion: Our data show that HMGB1-triggered joint inflammation is not mediated via the TNF pathway. Combined with our previous study, we suggest that HMGB1-triggered arthritis is probably mediated through IL-1 activation.

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Impact of high mobility group box chromosomal protein 1 on reactivity in the presence/absence of TNFα. Proliferative responses of splenocytes from TNFα+/+ mice and from TNFα-/- mice (n = five mice per group) incubated with different doses of (a) lipopolysaccharide-free purified recombinant endotoxin-free high mobility group box chromosomal protein 1 (pHMGB1) or (b) concavalin A (con A). Box plots, 25th and 75th percentiles; horizontal solid lines, medians; horizontal hatched lines, means; vertical bars, 5th and 95th percentiles. Statistical differences were calculated using the Mann–Whitney U test. CPM, counts per minute.
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Figure 2: Impact of high mobility group box chromosomal protein 1 on reactivity in the presence/absence of TNFα. Proliferative responses of splenocytes from TNFα+/+ mice and from TNFα-/- mice (n = five mice per group) incubated with different doses of (a) lipopolysaccharide-free purified recombinant endotoxin-free high mobility group box chromosomal protein 1 (pHMGB1) or (b) concavalin A (con A). Box plots, 25th and 75th percentiles; horizontal solid lines, medians; horizontal hatched lines, means; vertical bars, 5th and 95th percentiles. Statistical differences were calculated using the Mann–Whitney U test. CPM, counts per minute.

Mentions: To assess the impact of HMGB1 on the reactivity of immunocompetent cells in the presence or absence of the TNFα gene, spleen cells from TNFα+/+ mice and from knockout mice were stimulated with different concentrations of endotoxin-free pHMGB1 and the proliferative response was scored after 72 hours. Upon stimulation with the highest HMGB1 dose (5 μg/ml), TNFα+/+ mice had a significantly better proliferative response than their knockout littermates (285 ± 51 (median 264) counts per minute versus 197 ± 15 (median 185) counts per minute, respectively; P < 0.05), whereas no differences were seen regarding proliferation at lower HMGB1 concentrations (Figure 2). In addition, knockout mice had a threefold to fourfold lower response to concavalin A, a compound known to act on T lymphocytes, as compared with the TNFα+/+ mice (3,700 ± 246 (median 3,668) counts per minute versus 7,423 ± 1,043 (median 6,550) counts per minute, respectively; P = 0.009).


Induction of arthritis by high mobility group box chromosomal protein 1 is independent of tumour necrosis factor signalling.

Pullerits R, Jonsson IM, Kollias G, Tarkowski A - Arthritis Res. Ther. (2008)

Impact of high mobility group box chromosomal protein 1 on reactivity in the presence/absence of TNFα. Proliferative responses of splenocytes from TNFα+/+ mice and from TNFα-/- mice (n = five mice per group) incubated with different doses of (a) lipopolysaccharide-free purified recombinant endotoxin-free high mobility group box chromosomal protein 1 (pHMGB1) or (b) concavalin A (con A). Box plots, 25th and 75th percentiles; horizontal solid lines, medians; horizontal hatched lines, means; vertical bars, 5th and 95th percentiles. Statistical differences were calculated using the Mann–Whitney U test. CPM, counts per minute.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483464&req=5

Figure 2: Impact of high mobility group box chromosomal protein 1 on reactivity in the presence/absence of TNFα. Proliferative responses of splenocytes from TNFα+/+ mice and from TNFα-/- mice (n = five mice per group) incubated with different doses of (a) lipopolysaccharide-free purified recombinant endotoxin-free high mobility group box chromosomal protein 1 (pHMGB1) or (b) concavalin A (con A). Box plots, 25th and 75th percentiles; horizontal solid lines, medians; horizontal hatched lines, means; vertical bars, 5th and 95th percentiles. Statistical differences were calculated using the Mann–Whitney U test. CPM, counts per minute.
Mentions: To assess the impact of HMGB1 on the reactivity of immunocompetent cells in the presence or absence of the TNFα gene, spleen cells from TNFα+/+ mice and from knockout mice were stimulated with different concentrations of endotoxin-free pHMGB1 and the proliferative response was scored after 72 hours. Upon stimulation with the highest HMGB1 dose (5 μg/ml), TNFα+/+ mice had a significantly better proliferative response than their knockout littermates (285 ± 51 (median 264) counts per minute versus 197 ± 15 (median 185) counts per minute, respectively; P < 0.05), whereas no differences were seen regarding proliferation at lower HMGB1 concentrations (Figure 2). In addition, knockout mice had a threefold to fourfold lower response to concavalin A, a compound known to act on T lymphocytes, as compared with the TNFα+/+ mice (3,700 ± 246 (median 3,668) counts per minute versus 7,423 ± 1,043 (median 6,550) counts per minute, respectively; P = 0.009).

Bottom Line: HMGB1 is actively released from immune cells in response to TNFalpha; once released, HMGB1 in turn induces production of several proinflammatory cytokines--including IL-6 and TNFalpha--by macrophages.The purpose of the present study was to investigate whether the arthritis-inducing effect of HMGB1 is dependent on TNFalpha expression in vivo and to assess whether TNFalpha deficiency affects a proinflammatory cytokine response to HMGB1 in vitro.Our data show that HMGB1-triggered joint inflammation is not mediated via the TNF pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Rheumatology and Inflammation Research, Sahlgrenska Academy at Göteborg University, Guldhedsgatan 10A, 41346, Göteborg, Sweden. rille.pullerits@rheuma.gu.se

ABSTRACT

Introduction: TNFalpha and high mobility group box chromosomal protein 1 (HMGB1) are two potent proinflammatory cytokines implicated as important mediators of arthritis. Increased levels of these cytokines are found in the joints of rheumatoid arthritis patients, and the cytokines trigger arthritis when applied into the joints of naïve mice. HMGB1 is actively released from immune cells in response to TNFalpha; once released, HMGB1 in turn induces production of several proinflammatory cytokines--including IL-6 and TNFalpha--by macrophages. Whether HMGB1-induced arthritis is mediated via the TNFalpha pathway, however, is unknown. The purpose of the present study was to investigate whether the arthritis-inducing effect of HMGB1 is dependent on TNFalpha expression in vivo and to assess whether TNFalpha deficiency affects a proinflammatory cytokine response to HMGB1 in vitro.

Methods: TNFalpha knockout mice and backcrossed control animals on a C57Bl6 background were injected intraarticularly with 5 microg HMGB1. Joints were dissected 3 days after intraarticular injection and were evaluated histologically by scoring the frequency and severity of arthritis. For in vitro studies, mouse spleen cultures from TNFalpha knockout mice and from control mice were incubated with different doses of HMGB1, and cell culture supernatants were collected at different time points for analysis of IL-6.

Results: Intraarticular injection of HMGB1 into healthy mouse joints resulted in an overall frequency of 32% to 39% arthritic animals. No significant differences were found with respect to the severity and incidence of synovitis between mice deficient for TNFalpha (seven out of 18 mice with arthritis) in comparison with control TNFalpha+/+ animals (six out of 19). No significant differences were detected between spleen cells from TNFalpha+/+ mice versus TNFalpha-/- mice regarding IL-6 production upon stimulation with highly purified HMGB1 after 24 hours and 48 hours. Upon stimulation with a suboptimal dose of recombinant HMGB1, however, the splenocytes from TNFalpha+/+ animals released significantly more IL-6 than cells from the knockout mice (602 +/- 112 pg/ml and 304 +/- 50 pg/ml, respectively; P < 0.05).

Conclusion: Our data show that HMGB1-triggered joint inflammation is not mediated via the TNF pathway. Combined with our previous study, we suggest that HMGB1-triggered arthritis is probably mediated through IL-1 activation.

Show MeSH
Related in: MedlinePlus