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Cited by 66Stress Responses and CortisolNeuroinflammation and Neurodegeneration MechanismsAdrenal Hormones and Disorders

Interleukin-6 is an afferent signal to the hypothalamo-pituitary-adrenal axis during local inflammation in mice

Turnbull, Andrew V., Prehar, Sukhpal, Kennedy, Adam R., Little, Roderick A., Hopkins, Stephen J.·2003·Endocrinology
DOI 10.1210/en.2002-220964
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Abstract

The cytokines IL-1 and IL-6 are able to induce prostaglandin (PG)-dependent activation of the hypothalamo-pituitary-adrenal axis (HPAA) and are thought to play key roles in immune-neuroendocrine interactions during inflammation. The present study shows that inflammation induced by im injection of turpentine (TPS) in the hind limb of mice causes an increase in the plasma concentration of IL-6, but not that of IL-1α or IL-1β, together with a prolonged (>18-h) activation of the HPAA. IL-6 plays a causal role in the TPS-induced elevation in HPAA activity, because the sustained (8–18 h) increases in 1) plasma corticosterone, 2) plasma ACTH, and 3) induction of c-Fos in the hypothalamic paraventricular nucleus are all markedly blunted in IL-6-deficient (IL-6−/−) mice. Peripheral administration of a neutralizing IL-6 antiserum inhibited the plasma corticosterone response of normal (C57BL/6) mice to hind limb inflammation to an extent similar to that seen in IL-6−/− mice, suggesting that the IL-6 responsible for the increased HPAA activity is produced, or acts, on the blood side of the blood-brain barrier. We also show that IL-6 in the circulation is induced almost exclusively at the local inflammatory site, where IL-1β is produced. Induction of IL-6 and activation of the HPAA are dependent upon prior activation of an IL-1 type I receptor, as both are inhibited in type I IL-1 receptor-deficient mice. Furthermore, hind limb inflammation induced cyclooxygenase-2 protein expression around the cerebrovasculature of normal (IL-6+/+), but not IL-6−/−, mice. Based on these data, we propose that IL-6 is produced at the local inflammatory site under the control of IL-1β and is the circulating afferent signal that is in part responsible for elevated HPAA activity, possibly acting via eicosanoid production within the cerebrovasculature.

AI snapshot

Written from the paper’s full text.

TL;DR

In a mouse model of turpentine-induced local hind-limb inflammation, IL-6 is induced at the inflammatory site under IL-1 control and is required for the sustained hypothalamo-pituitary-adrenal axis (HPAA) response. The paper proposes IL-6 as a circulating afferent signal from inflamed tissue to the brain, possibly acting via cerebrovascular COX-2.

Key findings

  • Turpentine injection increased limb edema and plasma corticosterone rapidly, but plasma IL-6 appeared later, peaking at 8–12 h; plasma IL-1β was below detection (<28 pg/ml) and IL-1α did not increase significantly.
  • In IL-6-deficient mice, the sustained corticosterone response was blunted by 36% at 8 h, 73% at 12 h, and 46% at 18 h; with a lower 5-µl TPS dose at 12 h, corticosterone was reduced by 70% (256 ± 43 vs 77 ± 15 ng/ml).
  • At 12 h after TPS, plasma ACTH was 355 ± 71 pg/ml in IL-6+/+ mice versus 209 ± 23 pg/ml in IL-6−/− mice, and c-Fos in the ipsilateral PVN was significantly reduced in IL-6−/− mice.
  • Anti-IL-6 serum blunted the 10-h corticosterone response; IL-1RI−/− mice also had blunted corticosterone and no significant plasma IL-6 rise after TPS (saline 67 ± 34 pg/ml; TPS 143 ± 37 pg/ml) versus controls (saline 30 ± 0 pg/ml; TPS 1954 ± 150 pg/ml). Tourniquet occlusion of the inflamed limb reduced plasma IL-6 by over 90%, with half-life 1.29 ± 0.13 min, and COX-2 immunoreactivity around brain cerebrovasculature was present in IL-6+/+ but not IL-6−/− mice after TPS.

Methods

  • Male Sprague Dawley rats and female C57BL/6 mice, including IL-6−/−, IL-1RI−/−, and control strains, received intramuscular turpentine or saline; some mice underwent restraint stress.
  • Outcomes included plasma corticosterone by RIA, ACTH by immunoradiometric assay, plasma/tissue cytokines by bioassay or immunoassay, limb fluid content, c-Fos and COX-2 immunohistochemistry, anti-IL-6 immunoneutralization, and tourniquet occlusion of the inflamed limb in rats.
  • Data are presented as mean ± SEM and analyzed with paired/unpaired t tests or one-way ANOVA followed by Tukey-Kramer multiple comparisons, with P < 0.05 considered significant.

Limitations

  • The paper states that it does not exclude a direct IL-6 action on the pituitary or adrenal, does not claim plasma IL-6 is the only or most important HPAA activator, and notes that early HPAA activation is likely neurally mediated because plasma IL-6 appears after the corticosterone response begins.
  • The paper states that mechanisms beyond the blood-brain barrier remain unclear, including how IL-6 and prostaglandins signal to the CNS; COX-2 measurements were made in a single section per brain, though sections throughout the brain reportedly showed the same result.
  • Evident from the text: findings rely on mixed species and sexes, several cytokine measurements were near assay detection limits, and some comparisons used different control strains or limited group sizes.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1210/en.2002-220964

Cites

68 of 68 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 43 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    IL-6-deficient mice do not exhibit a lesser degree of hind limb swelling compared with controls after TPS injection.

    “However, Table 2 shows that mice without IL-6 did not exhibit a lesser degree of hind limb swelling compared with controls.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Limb edema is a valid measure of the degree of local inflammation.
  • Findingown data

    Peripheral administration of a neutralizing IL-6 antiserum inhibits the plasma corticosterone response of normal C57BL/6 mice to hind limb inflammation to an extent similar to that seen in IL-6-deficient mice.

    “Peripheral administration of a neutralizing IL-6 antiserum inhibited the plasma corticosterone response of normal (C57BL/6) mice to hind limb inflammation to an extent similar to that seen in IL-6 ؊/؊ mice, suggesting that the IL-6 responsible for the increased HPAA activity is produced, or acts, on the blood side of the blood-brain barrier.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The neutralizing IL-6 antiserum effectively neutralizes circulating IL-6 in vivo.
  • Findingown data

    The sustained (8-18 h) increases in plasma corticosterone, plasma ACTH, and c-Fos induction in the hypothalamic paraventricular nucleus after TPS are markedly blunted in IL-6-deficient mice.

    “IL-6 plays a causal role in the TPS-induced elevation in HPAA activity, because the sustained (8 -18 h) increases in 1) plasma corticosterone, 2) plasma ACTH, and 3) induction of c-Fos in the hypothalamic paraventricular nucleus are all markedly blunted in IL-6-deficient (IL-6 ؊/؊ ) mice.”
    Quote in the paperSubstantial claim
  • Findingown data

    IL-6-deficient mice have significantly lower plasma corticosterone concentrations than wild-type mice at 8-18 h after intramuscular TPS injection.

    “In marked contrast, the plasma corticosterone concentrations of IL-6-deficient mice were significantly (P Ͻ 0.01 to P Ͻ 0.05) lower than those of wild-type mice at 8 -18 h after im injection of TPS (Fig. 4).”
    Quote in the paperSubstantial claim
  • Findingown data

    There is no significant difference in plasma corticosterone between female IL-6+/+ and IL-6-/- mice that are untreated or saline-injected at least 8 h previously.

    “In each of a total of six experiments there was no significant difference (by one-way ANOVA followed by Tukey-Kramer multiple comparison test) between the plasma corticosterone concentrations of female IL-6 ϩ/ϩ mice (29.9 Ϯ 5.2; n ϭ 37) and IL-6 Ϫ/Ϫ mice (26.7 Ϯ 5.2; n ϭ 35) that had been either untreated or injected with saline at least 8 h previously.”
    Quote in the paperSubstantial claim
  • May combine two claims. 44
  • Classified as probably not a substantial claim. 25

  • May combine two claims.

    Findingown data

    COX-2 immunoreactivity is associated with the cerebrovasculature of IL-6+/+ mice 12 h after TPS injection, whereas no staining is seen in controls.

    “The results clearly showed immunoreactivity for COX-2 associated with the cerebrovasculature of IL-6 ϩ/ϩ mice 12 h after the injection of TPS (Fig. 11A), whereas no staining was seen in controls (not shown).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: COX-2 immunoreactivity in the cerebrovasculature indicates COX-2 induction.

  • May combine two claims.

    Findingown data

    Both IL-6 induction and HPAA activation after TPS-induced hind limb inflammation are inhibited in type I IL-1 receptor-deficient mice.

    “Induction of IL-6 and activation of the HPAA are dependent upon prior activation of an IL-1 type I receptor, as both are inhibited in type I IL-1 receptor-deficient mice.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Sections throughout the entire brain showed quantitatively the same result for COX-2 staining.

    “Although measurements were made in a single section of each brain, sections throughout the entire brain showed quantitatively the same result.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: COX-2 immunoreactivity in the cerebrovasculature indicates COX-2 induction.

  • May combine two claims.

    Findingown data

    Four hours after TPS injection, muscle homogenates from IL-6+/+ and IL-6-/- mice show increased IL-1β, IL-6, and TNFα, but no evidence of increased tissue IL-1α.

    “Homogenization of mus- cle collected 4 h after injection of TPS into IL-6 ϩ/ϩ and IL-6 Ϫ/Ϫ mice revealed that IL-1␤, IL-6, and TNF␣ were increased, although there was no evidence of an increase in tissue IL-1␣ (Table 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In normal mice, intramuscular injection of TPS into the hind limb induces local inflammation that is evident at 1 h, maximal by 8 h, and relatively well maintained at 36 h.

    “After im injection of TPS into the hind limb, inflammation was evident at 1 h, as judged by limb edema, and was maximal by 8 h (Fig. 1A).The limb edema remained relatively well maintained at 36 h.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After application of a tourniquet to the TPS-injected limb, plasma IL-6 falls rapidly, by over 90% at 10 min, with a half-life of 1.29±0.13 min.

    “In contrast, application of a tourniquet to the TPS-injected limb caused the plasma IL-6 concentration to fall rapidly, and by 10 min after tourniquet application plasma IL-6 fell by over 90% (half-life, 1.29 Ϯ 0.13 min).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    After TPS injection, IL-6 in the circulation is induced almost exclusively at the local inflammatory site, where IL-1β is produced.

    “We also show that IL-6 in the circulation is induced almost exclusively at the local inflammatory site, where IL-1␤ is produced.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Application of a tourniquet to the inflamed limb prevents IL-6 produced at the inflammatory site from entering the systemic circulation.

  • May combine two claims.

    Findingown data

    After intramuscular TPS injection, the ACTH response is rapid and remains significantly elevated at 12 h.

    “The ACTH response was also rapid (Fig. 2A) and remained significantly elevated at 12 h (Fig. 2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mice, inflammation induced by intramuscular injection of turpentine (TPS) in the hind limb causes an increase in plasma IL-6, but not plasma IL-1α or IL-1β, together with prolonged (>18 h) activation of the hypothalamo-pituitary-adrenal axis (HPAA).

    “The present study shows that inflammation induced by im injection of turpentine (TPS) in the hind limb of mice causes an increase in the plasma concentration of IL-6, but not that of IL-1␣ or IL-1␤, together with a prolonged (>18-h) activation of the HPAA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The increase in IL-1β after TPS injection is significantly greater in IL-6-/- mice, while the TNFα response is not significantly different.

    “The increase in IL-1␤ was actually significantly greater in IL-6 Ϫ/Ϫ mice, although the TNF␣ response was not significantly different.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After intramuscular TPS injection, the number of c-Fos-positive cells is higher in the contralateral PVN, but this is significant only at 12 h.

    “Interestingly, the number of c-Fos-positive cells was higher in the contralateral PVN, although this was only significant 12 h after injection of TPS.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: c-Fos immunoreactivity in the PVN indicates neuronal activation.

  • May combine two claims.

    Findingown data

    In normal mice, intramuscular TPS injection causes a significant corticosterone response at 1 h, peaking at 4 h and declining over the next 14 h.

    “The corticosterone response was also significant 1 h after the injection of TPS, peaking at 4 h and declining over the next 14 h (Fig. 1B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Preadministration of a neutralizing antirat IL-6 serum to C57BL/6 mice significantly blunts the corticosterone response at 10 h after TPS injection without affecting the inflammatory response.

    “Further evidence to support the hypothesis that IL-6 is an important contributor to HPAA activation during the inflammatory response was provided by the observation that preadministration of a neutralizing antirat IL-6 serum to C57BL/6 mice was able to blunt the corticosterone response significantly at 10 h (Fig. 5), while again not affecting the inflammatory response (Table 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In IL-6-/- mice, the number of c-Fos-positive cells in the PVN is reduced, but this is significant only on the ipsilateral side (P<0.05).

    “However, in IL-6 Ϫ/Ϫ mice the number of c-Fos-positive cells was reduced, although this was significant only on the ipsilateral side of the PVN (Fig. 8; P Ͻ 0.05).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: c-Fos immunoreactivity in the PVN indicates neuronal activation.

  • May combine two claims.

    Findingown data

    Corticosterone responses of IL-6-deficient mice after TPS injection are inhibited by 36% at 8 h, 73% at 12 h, and 46% at 18 h.

    “Corticosterone responses of IL-6deficient mice were inhibited by 36% at 8 h, 73% at 12 h, and 46% at 18 h.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In normal mice, intramuscular TPS injection causes increased plasma IL-6 that is detected only after 4 h, peaks at 8-12 h, and remains raised until at least 18 h.

    “Increased plasma IL-6 was detected only after 4 h, peaking at 8 -12 h and remaining raised until at least 18 h (Fig. 1C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After TPS injection, c-Fos staining in the PVN increases on both the contralateral and ipsilateral sides of both IL-6+/+ and IL-6-/- mice, with staining more marked on the contralateral side.

    “Examination of c-Fos staining in the PVN revealed that the number of activated cells increased on both the contralateral and ipsilateral sides of both IL-6 ϩ/ϩ and IL-6 Ϫ/Ϫ mice, although the staining was more marked on the contralateral side (Table 1 and Fig. 7B).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: c-Fos immunoreactivity in the PVN indicates neuronal activation.

  • May combine two claims.

    Findingown data

    In IL-6-/- mice, 12 h after TPS injection, plasma ACTH concentrations (209±23 pg/ml) do not differ significantly from saline-treated controls of either genotype but are significantly lower than those in TPS-treated IL-6+/+ mice.

    “In contrast, 12 h after induction of local inflammation in IL-6 Ϫ/Ϫ mice, plasma ACTH concentrations (209 Ϯ 23 pg/ml) did not differ significantly from saline-treated controls of either genotype, but were significantly (P Ͻ 0.05) lower than those in TPS-treated IL-6 ϩ/ϩ mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The corticosterone response of IL-1RI-/- mice is significantly blunted relative to controls, whereas local inflammation at 12 h is not affected.

    “The importance of IL-1 in the overall response is shown by the fact that the corticosterone response of IL-1RI Ϫ/Ϫ mice is, like that of IL-6 Ϫ/Ϫ mice, significantly blunted relative to the control value (Fig. 9), whereas local inflammation at 12 h is not affected (Table 5).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: IL-1RI-/- mice lack functional IL-1 type I receptor signaling.

  • May combine two claims.

    Findingown data

    We found no evidence for an increase in the IL-1 concentration in the plasma of mice injected with TPS despite a significant local IL-1β response in the tissue.

    “However, we found no evidence for an increase in the IL-1 concentration in the plasma of mice injected with TPS despite a significant local IL-1␤ response in the tissue.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The acute HPAA response to both noninflammatory stress and inflammation is independent of IL-6.

    “mediated) response to both noninflammatory stress and inflammation is independent of IL-6.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Plasma ACTH measured from decapitated animals was inconsistent and unreliable, so ACTH was measured from cardiac puncture of anesthetized mice.

    “We found that plasma obtained from decapitated animals assayed for ACTH produced inconsistent and unreliable data, and plasma samples did not dilute parallel to the standard curve.Consequently, plasma ACTH concentrations were measured on plasma derived from a blood sample obtained by cardiac puncture of mice anesthetized with a high dose (100 l of 30 mg/ml) of sodium pentobarbital per mouse.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    IL-6 is produced at the local inflammatory site under the control of IL-1β and is the circulating afferent signal that is in part responsible for elevated HPAA activity, possibly acting via eicosanoid production within the cerebrovasculature.

    “Based on these data, we propose that IL-6 is produced at the local inflammatory site under the control of IL-1␤ and is the circulating afferent signal that is in part responsible for elevated HPAA activity, possibly acting via eicosanoid production within the cerebrovasculature.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Within the range of values measured, recovery of exogenous recombinant cytokines was between 85% and 100%.

    “Within the range of values measured, recovery of exogenous recombinant cytokines was between 85% and 100%.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The cross-reaction of the antirat IL-6 antiserum was apparent with respect to its efficacy in immunoassays of both rat and mouse IL-6.

    “The cross-reaction of this antiserum was apparent with respect to its efficacy in immunoassays of both rat and mouse IL-6.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    Controls for IL-1R-/- mice were C57BL/6 x C57J/129, the appropriate control strain cross.

    “Controls for IL-1R Ϫ/Ϫ mice were C57BL/6 ϫ C57J/129 (the appropriate control strain cross).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Experiments were performed such that sample collection was at a similar time of day (0900-1100 h) to minimize variations due to circadian factors.

    “Experiments were performed such that the collection of samples was performed at a similar time of day (0900 -1100 h) to minimize variations due to circadian factors.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The interassay coefficient of variation for the IL-6 assay was less than 12% within the assay range.

    “The interassay coefficient of variation for the IL-6 assay was less than 12% within the assay range.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Within- and between-assay coefficients of variation for ACTH at low and high concentrations were less than 10%, and all plasma ACTH concentrations from a single experiment were measured in the same assay.

    “Within-and between-assay coefficients of variation at low (50 pg/ml) and high (350 pg/ml) concentrations were less than 10%, and all plasma ACTH concentrations from a single experiment were measured in the same assay.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The pain and stress elements of inflammation are IL-6 independent.

    “As pain causes stress, and we have demonstrated that stress-induced HPAA activation is IL-6 independent, it is not surprising that the pain and stress elements of inflammation should be IL-6 independent.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Pain causes stress.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The present experiments do not exclude the possibility that IL-6 may have contributed to the pituitary or adrenal response by a direct action on these tissues.

    “The present experiments do not exclude the possibility that IL-6 may have contributed to the pituitary or adrenal response by a direct action on these tissues, and such effects have certainly been shown (8).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] Turnbull, 1999, Regulation of the hypothalamic-pituitary-adrenal axis by cytokines: actions and mechanisms of action., Physiol Rev source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data
    § Interleukin-6 Is an Afferent Signal to the Hypothalamo-Pituitary-Adrenal Axis during Local Inflammation in Mice

    IL-6, induced by IL-1 at the site of inflammation, is the key cytokine involved in systemic mediation of the signal from the tissues to the HPAA and is required for induction of COX-2 in the brain microvasculature.

    “We found that IL-6, induced by IL-1 at the site of inflammation, is the key cytokine involved in systemic mediation of the signal from the tissues to the HPAA and is required for induction of cyclooxygenase-2 (COX-2) in the brain microvasculature.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: IL-1 is a potent inducer of IL-6.

  • May combine two claims.

    Interpretationown data

    The efficacy of IL-6 neutralization suggests that deficient corticosterone responsiveness is unlikely to be attributable to a developmental effect in IL-6-/- mice and that the IL-6 inducing the HPAA response is probably at least produced, even if it does not act, on the blood side of the blood-brain barrier.

    “The efficacy of IL-6 neutralization suggests that deficient corticosterone responsiveness is unlikely to be attributable to any developmental effect in IL-6 Ϫ/Ϫ mice and that the IL-6 inducing the HPAA response is probably at least produced, even if it does not act, on the blood side of the blood-brain barrier.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    IL-6 dependency of the HPAA response is not sex dependent.

    “Our experiments incidentally show that IL-6 dependency is not sex dependent.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Preadsorbing the anti-c-Fos antiserum with synthetic c-Fos peptide immunogen abolished specific staining.

    “Preadsorbing the anti-c-Fos antiserum overnight at 4 C with 10 g synthetic c-Fos peptide immunogen abolished specific staining.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    Assay of plasma samples derived by cardiac puncture produced consistent ACTH values and diluted in parallel to the standard curve.

    “Assay of plasma samples so derived produced consistent ACTH values, and these samples diluted in parallel to the standard curve.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    All intraassay coefficients of variation for other cytokine comparisons were less than 10%.

    “Other cytokine comparisons were determined from measurements within a single assay, and all intraassay coefficients of variation were less than 10%.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    In mice without CRF type 1 receptors, both corticosterone and ACTH were still increased in response to TPS.

    “In mice without CRF type 1 receptors, both corticosterone and ACTH were still increased in response to TPS (10).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Turnbull, 1999, CRF type I receptor-deficient mice exhibit a pronounced pituitary-adrenal response to local inflammation., Endocrinology source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Early HPAA activation after TPS-induced inflammation cannot be induced by plasma IL-6 because plasma IL-6 appeared after the corticosterone response was initiated.

    “Early activation clearly cannot be induced in this way, because plasma IL-6 appeared after the corticosterone response was initiated.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    Tourniquet application resulted in the limb turning blue within 30 sec, indicating that blood flow to and from the inflammatory site had ceased.

    “Tourniquet application resulted in the limb turning blue within 30 sec, indicating that blood flow to (and therefore from) the inflammatory site had ceased.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The IL-6 responsible for increased HPAA activity after TPS-induced hind limb inflammation is produced, or acts, on the blood side of the blood-brain barrier.

    “Peripheral administration of a neutralizing IL-6 antiserum inhibited the plasma corticosterone response of normal (C57BL/6) mice to hind limb inflammation to an extent similar to that seen in IL-6 ؊/؊ mice, suggesting that the IL-6 responsible for the increased HPAA activity is produced, or acts, on the blood side of the blood-brain barrier.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The neutralizing IL-6 antiserum effectively neutralizes circulating IL-6 in vivo.

  • Classified as probably not a substantial claim.

    Premisecitation

    The timing of serum injections (10 h before sampling) was determined from previous descriptions of temporal profiles of antibody titers in plasma after intraperitoneal administration of antisera to rats and from successful immunoneutralization experiments in mice.

    “The timing of serum injections (10 h before sampling time) was determined from previous descriptions of the temporal profiles of antibody titers in plasma after ip administration of antisera to rats (14) and from successful immunoneutralization experiments in mice (10).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [14] Turnbull, 1998, Intracerebroventricular passive immunization. II. Intracerebroventricular infusion of neuropeptide antisera can inhibit neuropeptide signaling in peripheral tissues., Endocrinology source

      Not checked in the free run.

    • cites [10] Turnbull, 1999, CRF type I receptor-deficient mice exhibit a pronounced pituitary-adrenal response to local inflammation., Endocrinology source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Specific COX-2 staining was abolished by preadsorbing the anti-COX-2 antibody with synthetic peptide immunogen.

    “Specific staining was abolished by preadsorbing the anti-COX-2 antibody overnight at 4 C with 50 g synthetic peptide immunogen.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Induction of IL-6 and activation of the HPAA after TPS-induced hind limb inflammation are dependent upon prior activation of an IL-1 type I receptor.

    “Induction of IL-6 and activation of the HPAA are dependent upon prior activation of an IL-1 type I receptor, as both are inhibited in type I IL-1 receptor-deficient mice.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Some experiments have shown that IL-6 alone is unable to induce PGs in brain microvessels.

    “However, some experiments have shown that IL-6 alone is unable to induce PGs in brain microvessels (62).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [62] Bishai, 1996, Differential-effects of endotoxin and cytokines on prostaglandin E2 formation in cerebral microvessels and brain parenchyma: implications for the pathogenesis of fever., Cytokine source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    Inflammation and IL-1 induce the production of soluble IL-6R (sIL-6R), which binds, but does not inactivate, IL-6.

    “Thirdly, inflammation and IL-1 induce the production of soluble IL-6R (sIL-6R), which binds, but does not inactivate, IL-6.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IL-6R augments the central effects of IL-6 on several CNS-regulated parameters.

    “It has previously been shown that IL-6R augments the central effects of IL-6 on several CNSregulated parameters (51).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [51] Schöbitz, 1995, Soluble interleukin-6 (IL-6) receptor augments central effects of IL-6 in vivo., FASEB J source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Early glucocorticoid responses may facilitate later IL-6-mediated regulation of the HPAA.

    “Interestingly, glucocorticoids themselves are able to increase the expression of IL-6R (52), which suggests that early glucocorticoid responses may facilitate later IL-6-mediated regulation of the HPAA.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] Fischer, 1996, Glucocorticoid-dependent induction of interleukin-6 receptor expression in human hepatocytes facilitates interleukin-6 stimulation of amino acid transport., Ann Surg source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The IL-6:sIL-6R complex has an increased affinity for gp130 and allows it to bind and activate cells, such as endothelium, that have no IL-6R themselves.

    “The IL-6:sIL-6R complex has an increased affinity for the signal transducing accessory receptor glycoprotein (gp130), and allows it to bind and activate cells, such as those of the endothelium, that have no IL-6R themselves (50).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [50] Romano, 1997, Role of IL-6 and its soluble receptor in induction of chemokines and leucocyte recruitment., Immunity source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Our data identify IL-1 as an important tissue cytokine responsible for induction of IL-6, which then acts as the systemic humoral mediator of HPAA activation.

    “Our data identify IL-1 as an important tissue cytokine, responsible for induction of the IL-6, which then acts as the systemic humoral mediator of HPAA activation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    TNFα is important in both the increase in plasma IL-6 and HPAA activation induced by TPS.

    “We have shown previously that TNF␣ is important in both the increase in plasma IL-6 and HPAA activation induced by TPS (11,42).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [11] Cooper, 1994, Tumour necrosis factor-α and fever after peripheral inflammation in the rat, Am J Physiol

      Not checked in the free run.

    • cites [42] Turnbull, 1997, Inhibition of tumor necrosis factor-α action within the CNS markedly reduces the plasma adrenocorticotropin response to peripheral local inflammation in rats., J Neurosci source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IL-6 is generally accepted to be less efficacious than IL-1 or TNFα in increasing fever and activating the HPAA.

    “Although IL-6 has clearly been shown to increase fever and activate the HPAA, it is generally accepted to be less efficacious than IL-1 or TNF␣ (45,46).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [45] Besedovsky, 1991, Cytokines as modulators of the hypothalamus-pituitary-adrenal axis., J Steroid Biochem Mol Biol source

      Not checked in the free run.

    • cites [46] Van der Meer, 1996, Acute stimulation of the hypothalamic-pituitary-adrenal axis by IL-1β, TNFα and IL-6: a dose response study., J Endocrinol Invest source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    IL-6 does not induce the production of additional circulating inflammatory mediators, whereas IL-1 induces sustained production of IL-6.

    “Secondly, IL-6 does not induce the production of additional circulating inflammatory mediators, whereas IL-1 induces sustained production of IL-6 (47).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Libert, 1990, Induction of interleukin-6 by human and murine recombinant interleukin-1 in mice, Eur J Immunol source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PG receptor subtypes appear to be differentially expressed in different regions of the brain, and this rather than the site of PG expression may be a critical factor.

    “However, PG receptor subtypes appear to be differentially expressed in different regions of the brain, and this, rather than the site of PG expression, may be a critical factor (63).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [63] Zhang, 1999, Distribution, regulation and colocalization of the genes encoding the EPl2- and EPl4-PGEl2 receptors in the brain and neuronal responses to systemic inflammation., Eur J Neurosci source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    IL-1β is important for the induction of IL-6 following induction of tissue inflammation by TPS, although it is not critical after systemic activation by LPS.

    “However, IL-1␤ is important for the induction of IL-6 following the induction of tissue inflammation by TPS, although it is not critical after systemic activation by LPS (21).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Fantuzzi, 1996, The inflammatory response in interleukin-1β-deficient mice: comparison with other cytokine-related knock-out mice., J Leukocyte Biol source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Glucocorticoids are able to increase the expression of IL-6R.

    “Interestingly, glucocorticoids themselves are able to increase the expression of IL-6R (52), which suggests that early glucocorticoid responses may facilitate later IL-6-mediated regulation of the HPAA.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] Fischer, 1996, Glucocorticoid-dependent induction of interleukin-6 receptor expression in human hepatocytes facilitates interleukin-6 stimulation of amino acid transport., Ann Surg source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Both IL-1 and IL-6 induce PGs in hypothalamic explants.

    “Both IL-1 and IL-6 induce PGs in hypothalamic explants (60), and arachidonate products are involved in IL-6-induction of CRF release (61).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [60] Navarra, 1992, Interleukin-1β and interleukin-6 specifically increase the release of prostaglandin E2 from rat hypothalamic explants in vitro., Neuroendocrinology source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Increased COX-2 expression in brain endothelium after injection of TPS has been shown.

    “However, further work has shown increased COX-2 expression in brain endothelium after the injection of TPS (56).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [56] Lacroix, 1998, Effect of acute systemic inflammatory response and cytokines on the transcription of the genes encoding cyclooxygenase enzymes (COX-1 and COX-2) in the rat brain., J Neurochem source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PG synthesis is important not only in the mediation of fever, but for other CNS responses as well, including HPAA activation.

    “Some clues are provided by data showing PG synthesis to be important not only in the mediation of fever, but for other CNS responses as well, including HPAA activation (5).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] Turnbull, 1996, Corticotropin-releasing factor, vasopressin, and prostaglandins mediate, and nitric oxide restrains, the hypothalamic-pituitary-adrenal response to acute local inflammation in the rat., Endocrinology source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    A relatively high dose of peripheral IL-6 produced a short-lasting induction of the HPAA, but was not effective when given intracerebroventricularly.

    “Wang and Dunn (49) also showed that, although less potent than IL-1␤, a relatively high dose of peripheral IL-6 produced a shortlasting induction of the HPAA, but it was not effective when given intracerebroventricularly.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [49] Wang, 1998, Mouse interleukin-6 stimulates the HPA axis and increases brain tryptophan and serotonin metabolism, Neurochem Int source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Constant infusion of IL-6 has been shown to induce c-Fos in the PVN, and this is COX-2 dependent.

    “In this respect, constant infusion of IL-6 has been shown to induce c-Fos in the PVN, and this is COX-2 dependent (48).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] Niimi, 1997, Effect of continuous intravenous injection of interleukin-6 and pretreatment with cyclooxygenase inhibitor on brain c-fos expression in the rat., Neuroendocrinology source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    IL-6 is apparently unable to induce PGs in peripheral mononuclear cells, although IL-6-induced fever is blocked by COX inhibitors, and IL-6 induces PGs in cerebrospinal fluid.

    “IL-6 is apparently unable to induce PGs in peripheral mononuclear cells, although IL-6-induced fever is blocked by COX inhibitors, and IL-6 induces PGs in cerebrospinal fluid (59).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [59] Dinarello, 1991, Interleukin-6 as an endogenous pyrogen: induction of prostaglandin-E2 in brain but not in peripheral blood mononuclear cells., Brain Res source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    IL-6 has clearly been shown to increase fever and activate the HPAA.

    “Although IL-6 has clearly been shown to increase fever and activate the HPAA, it is generally accepted to be less efficacious than IL-1 or TNF␣ (45,46).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [45] Besedovsky, 1991, Cytokines as modulators of the hypothalamus-pituitary-adrenal axis., J Steroid Biochem Mol Biol source

      Not checked in the free run.

    • cites [46] Van der Meer, 1996, Acute stimulation of the hypothalamic-pituitary-adrenal axis by IL-1β, TNFα and IL-6: a dose response study., J Endocrinol Invest source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IL-1 was the first cytokine clearly shown to have a direct action on the HPAA at the level of either the pituitary or the hypothalamus.

    “IL-1 was the first cytokine clearly shown to have a direct action on the HPAA at the level of either the pituitary (34) or the hypothalamus (35,36).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [34] Bernton, 1987, Release of multiple hormones by a direct action of interleukin-1 on pituitary cells., Science source

      Not checked in the free run.

    • cites [35] Sapolsky, 1987, Interleukin-1 stimulates the secretion of hypothalamic corticotrophin-releasing factor., Science source

      Not checked in the free run.

    • cites [36] Berkenbosch, 1987, Corticotropin-releasing factor-producing neurons in the rat activated by interleukin-1., Science source

      Not checked in the free run.

64 of 64

  • The paper relies on this without support.

    IL-6 is the only cytokine consistently observed to be increased in plasma during inflammation.

    “Because IL-6 is the only one of these cytokines consistently observed to be increased in plasma during inflammation, we sought to test the hypothesis that IL-6 is an important afferent signal in this situation.”

  • The paper relies on this without support.

    IL-1 is a potent inducer of IL-6.

    “IL-1 is a potent inducer of IL-6, and it seemed likely that it would be important at the site of inflammation.”

  • The paper relies on this without support.

    TPS-induced tissue inflammation is a valid model that corresponds closely to tissue injury and inflammation and avoids complex questions related to where LPS may be acting.

    “We investigated the role of IL-6 in a model of turpentine (TPS)-induced tissue inflammation, which is of particular value in corresponding more closely to tissue injury and inflammation, and avoids complex questions related to where lipopolysaccharide (LPS) may be act-ing.”

  • The paper relies on this without support.

    Limb edema is a valid measure of the degree of local inflammation.

    “The reduced corticosterone responses in IL-6 Ϫ/Ϫ mice from 8 h and beyond could not be attributed to a difference in the inflammatory response as determined by limb edema.”

  • The paper relies on this without support.

    c-Fos immunoreactivity in the PVN indicates neuronal activation.

    “Activation at the level of the hypothalamus was similarly apparent at 1 and 12 h, as indicated by the increase in the number of c-Fos-positive cells in the PVN (Table 1).”

  • The paper relies on this without support.

    COX-2 immunoreactivity in the cerebrovasculature indicates COX-2 induction.

    “The results clearly showed immunoreactivity for COX-2 associated with the cerebrovasculature of IL-6 ϩ/ϩ mice 12 h after the injection of TPS (Fig. 11A), whereas no staining was seen in controls (not shown).”

  • The paper relies on this without support.

    IL-1RI-/- mice lack functional IL-1 type I receptor signaling.

    “The IL-1 type I receptor-deficient (IL-1R Ϫ/Ϫ ) mice were obtained courtesy of Dr. John Simms (Immunex Corp., Seattle, WA).”

  • The paper relies on this without support.

    Pain causes stress.

    “As pain causes stress, and we have demonstrated that stress-induced HPAA activation is IL-6 independent, it is not surprising that the pain and stress elements of inflammation should be IL-6 independent.”

  • The paper relies on this without support.

    Application of a tourniquet to the inflamed limb prevents IL-6 produced at the inflammatory site from entering the systemic circulation.

    “A tourniquet was applied in the inguinal region of either the left (n ϭ 3) or the right (n ϭ 3) limb, and blood samples were taken at 1, 2, 5, 10, and 20 min.”

  • The paper relies on this without support.

    The neutralizing IL-6 antiserum effectively neutralizes circulating IL-6 in vivo.

    “Peripheral administration of a neutralizing IL-6 antiserum inhibited the plasma corticosterone response of normal (C57BL/6) mice to hind limb inflammation to an extent similar to that seen in IL-6 ؊/؊ mice, suggesting that the IL-6 responsible for the increased HPAA activity is produced, or acts, on the blood side of the blood-brain barrier.”

  • The paper relies on this without support.

    c-Fos expression in the hypothalamic paraventricular nucleus indicates neuronal activation.

    “IL-6 plays a causal role in the TPS-induced elevation in HPAA activity, because the sustained (8 -18 h) increases in 1) plasma corticosterone, 2) plasma ACTH, and 3) induction of c-Fos in the hypothalamic paraventricular nucleus are all markedly blunted in IL-6-deficient (IL-6 ؊/؊ ) mice.”
11 of 11

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Tourniquet sampling time differs between methods and figure

    Minor

    The methods say blood was sampled 3 minutes after tourniquet application, whereas the Fig. 10 legend lists 2 minutes instead.

  • Sex of mice in the IL-1 receptor comparison conflicts

    Minor

    The discussion says only female IL-1RI-deficient mice were available for the comparison, but the Fig. 9 legend identifies the IL-1-RI-deficient mice in that comparison as male.

2 of 2

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Related

Authors
Turnbull, A. V., Prehar, S., Kennedy, A. R., Little, R. A., & Hopkins, S. J.
Affiliations
  • University of Manchester (GB)
  • Salford Royal Hospital (GB)
Corresponding author
Hopkins, Stephen J.
ORCID
Venue
Endocrinology
Publisher
The Endocrine Society
Published
2003-05-01
Volume
144
Issue
5
Pages
1894-1906
ISSN
  • 0013-7227 (ISSN-L)
  • 1945-7170
DOI
10.1210/en.2002-220964
OpenAlex
W2050816700
Semantic Scholar
e4b522d15b300b8c2501e463803e98c1bd341ff2
Cited by
66
References
72
Citations by year
2025: 3 · 2023: 2 · 2021: 1 · 2020: 1 · 2019: 1 · 2018: 2 · 2017: 1 · 2016: 1 · 2015: 2 · 2014: 2 · 2013: 5 · 2012: 2
FWCI
2.26
Type
journal article
Language
en
Open access
closed
Locations
  • Endocrinology (publishedVersion)
  • PubMed (publishedVersion)
  • Research Explorer (The University of Manchester) (submittedVersion)
Funders
  • Johns Hopkins University
  • Medical Research Council
Topics
  • Life Sciences › Neuroscience › Behavioral Neuroscience › Stress Responses and Cortisol
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
Keywords
interleukin-6, hypothalamic-pituitary-adrenal axis, local inflammation, turpentine-induced inflammation, corticosterone, ACTH, paraventricular nucleus, c-fos, IL-1β, cyclooxygenase-2, eicosanoids, cerebral vasculature, neuroendocrine-immune interaction, afferent signals, blood-brain barrier
MeSH terms
Animals, Cerebrovascular Circulation, Endothelium, Vascular, Enzyme Induction, Female, Hindlimb, Hypothalamo-Hypophyseal System, Inflammation, Interleukin-1, Irritants, Isoenzymes, Male, Mice, Inbred C57BL, Pituitary-Adrenal System, Prostaglandin-Endoperoxide Synthases, Turpentine, Signal Transduction, Interleukin-6, Rats, Sprague-Dawley, Mice, Knockout, Mice, Rats, Cyclooxygenase 2

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

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