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Keywords:

  • Interleukin-1;
  • leptin;
  • microglia;
  • neuroinflammation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Leptin regulates energy balance by suppressing appetite and increasing energy expenditure through actions in the hypothalamus. Recently we demonstrated that the effects of leptin are, at least in part, mediated by the release of interleukin (IL)-1β in the brain. Microglia constitute the major source of IL-1β in the brain but it is not known whether these cells express leptin receptors, or respond to leptin to produce IL-1β. Using RT-PCR and immunocytochemistry, we demonstrate that primary rat microglial cells express the short (non-signalling) and long (signalling) isoforms of the leptin receptors (Ob-R)s. Immunoassays performed on cell medium collected 24 h after leptin treatment (0.01–10 μg/mL) demonstrated a dose-dependent production and release of IL-1β and its endogenously occurring receptor antagonist IL-1RA. In addition leptin-induced IL-1β release occurs via a signal transducer and activator of transcription 3 (STAT3)-dependent mechanism. Western blot analysis demonstrated that leptin induced the synthesis of pro-IL-1β in microglial cells and the release of mature 17 kDa isoform into the culture medium. Leptin-induced IL-1β release was neither inhibited by the pan-caspase inhibitor BOC-D-FMK, nor by the caspase 1 inhibitor Ac-YVAD-CHO indicating that IL-1 cleavage is independent of caspase activity. These results confirm our earlier observations in vivo and demonstrate that microglia are an important source of IL-1β in the brain in response to leptin.

Abbreviations used
IFNγ

interferon-γ

IL-1

interleukin-1

IL-6

interleukin-6

IL-1R1

IL-1 type 1 receptor

IL-1RA

interleukin-1 receptor antagonist

Ob-R

leptin receptor

STAT3

signal transducer and activator of transcription 3

Leptin is a 16 kDa product of the ob gene produced mainly by adipose tissue and released into the circulation (Zhang et al. 1994). This hormone is best known for its action as an afferent adiposity signal to the brain that suppresses appetite and increases energy expenditure (Friedman and Halaas 1998). Leptin also acts on peripheral targets to regulate immune function (Matarese et al. 2005). Leptin induces proliferation, differentiation, and functional activation of all types of haematopoietic cells (Gainsford et al. 1996) via binding to specific leptin receptors (Ob-Rs) and the activation of signal transducer and activator of transcription 3 (STAT3), Ras and phosphatidylinositol-3 kinase signalling pathways (Shanley et al. 2002; Bates et al. 2003; Durakoglugil et al. 2005). Macrophages from leptin-deficient (ob/ob) mice, show impaired phagocytic function (Loffreda et al. 1998; Lee et al. 1999). Leptin also stimulates production of typical pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6 and tumor necrosis factor-α in macrophages and circulating monocytes from normal animals and humans in vitro (Zarkesh-Esfahani et al. 2001; Dixit et al. 2004), and contributes to the development of a variety of inflammatory responses in vivo (Busso et al. 2002; Mancuso et al. 2002; Ikejima et al. 2005).

Interleukin-1 is a major mediator of inflammation, exerting a wide range of effects on the immune, endocrine and central nervous systems (CNS). IL-1β, the main released form of IL-1, exists as an inactive precursor molecule which requires cleavage by the enzyme caspase 1 into its biologically active ‘mature’ form. All actions of IL-1 are inhibited by a naturally occurring receptor antagonist (IL-1RA), which blocks IL-1 binding to its signalling receptor (Dinarello 1997). The activation of IL-1 signalling in the brain is an important regulator of systemic host defense responses to infection and inflammation including suppression of food intake and fever (e.g. increased thermogenesis) (Horai et al. 1998; Josephs et al. 2000).

Under normal, physiological conditions, brain IL-1 levels are extremely low and in most cases undetectable (Vitkovic et al. 2000), suggesting that this cytokine contributes little to physiological functions regulated by the brain. Recent evidence, however, suggests that, despite its low-expression, IL-1 could play a role in the homeostatic regulation of body weight and/or fat metabolism. IL-1RA deficient mice exhibit a lean phenotype and are resistant to diet-induced obesity when compared with their wild type controls, presumably due to enhanced/unchecked activity of IL-1 in the absence of the antagonist (Irikura et al. 2002; Matsuki et al. 2003; Somm et al. 2005). Conversely, mice lacking the IL-1R1 gene develop mature onset obesity (Garcia et al. 2006). Our own studies (Luheshi et al. 1999) and those of others (Garcia et al. 2006) demonstrated that the same mice (IL-1R1 knockouts) are resistant to the appetite suppressing effects of leptin. We also showed that administration of IL-1RA into the brain of normal rats abolishes the anorexic effect of leptin (Luheshi et al. 1999). We further found that neutralization of endogenous leptin by anti-leptin antiserum attenuated the increase of IL-1β mRNA expression in the hypothalamus, which was accompanied by reversal of the anorexia resulting from systemic inflammation induced by bacterial lipopolysaccharide (LPS) (Sachot et al. 2004). Collectively, these data indicate that the interaction between leptin and the IL-1 system in the brain plays an important role in body weight homeostasis both under physiological and inflammatory conditions. However, specific brain targets and cellular mechanisms of this interaction are largely unknown. As leptin exerts profound effects on peripheral immune cells, and microglia are the key immune cells in the CNS, we hypothesized that microglial cells are important target of leptin in the brain. Here we show that leptin induces the production and release of mature IL-1β and IL-1RA proteins in primary culture of rat microglia, and that the maturation of IL-1β by leptin is caspase 1 independent via an as yet unidentified mechanisms.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell cultures

Primary mixed glial cultures, and secondary cultures of astrocytes and microglia were prepared from brains of post-natal day 0–2 Sprague–Dawley rat (Charles River, Sandwich, UK) using a protocol previously described (Pinteaux et al. 2002). Primary cortical neuronal cell cultures were prepared from day 18 Sprague–Dawley rat embryos as previously described (Moore et al. 2002).

Reverse transcriptase-polymerase chain reaction

Total RNA was extracted from cell cultures using TRIzol reagent (Invitrogen, Paisley, UK). One μg of RNA was reverse transcripted using Moloney Murine Leukemia Virus (Invitrogen, Paisley, UK) according to manufactures instructions. PCR was carried out with Biotaq DNA polymerase, contained in a ready-made two times reaction mix (BioMixRed) (Bioline, London, UK). Specific oligonucleotide primers, annealing temperatures and cycle numbers are as follows: Ob-Ra, forward, 5′-ACACTGTTAATTTCACACCAGAG-3′, reverse, 5′-AGTCATTCAAACCATAGTTTAGG-3′, 59°C and 40 cycle; Ob-Rb, forward, 5′-ACACTGTTAATTTCACACCAGAG-3′, reverse, 5′-TTCCAAAAGCTCATCCAACCC-3′, 59°C and 40 cycle; and β-actin, forward, 5′-GCCGTCTTCCCCTCCATCGTG-3′, reverse, 5′-TACGACCAGAGGCATACAGGGACAAC-3′, 60°C and 22 cycle. For all experiments, control reactions using total RNA were performed to ensure that amplification was not a result of contamination with genomic DNA.

Immunocytochemistry

Microglial cells seeded on glass cover slips were stained by incubation of living cells with a Fluorescein-isothiocyanate-conjugated Lectin Griffonia Simplicifolia (GSL)-IB4 (Vector, Burlingame, UK) (1 : 400 dilution) for 30 min at room temperature (22–25°C). Cells were washed and Ob-R immunostaining was then carried out as previously described (Pinteaux et al. 2002), using an IgG anti-leptin receptor (Ob-R) primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1 : 500 dilution) in absence or presence of an excess (10 μg/mL) of recombinant Ob-R blocking peptide (Santa Cruz Biotechnology), and subsequent incubation with a Texas-Red-conjugated affinity-purified donkey anti-goat IgG (Chemicon, Temecula, CA, USA) (1 : 50 dilution).

Treatments

Cells were exposed to normal culture medium (Control) or treated for 24 h with increasing concentrations (0.01, 0.1, 0.5, 1, 5 or 10 μg/mL) of recombinant mouse leptin provided by Dr Stephen Poole from the National Institute for Biological Standards and Control (NIBSC, Potters Bar, UK) or rat leptin (Peprotech, London, UK) diluted in phosphate buffered saline. To ensure that the IL-1β release was not due to endotoxin contamination, cells were incubated with heat-inactivated (95°C, 30 min) leptin (10 μg/mL) for 24 h. STAT3 activation was carried out by incubating cells with leptin (10 μg/mL) for 5, 15, 30 or 60 min, or with IFNγ (100 IU/mL) for 30 min. Some cells were pre-treated with a specific STAT3 peptide inhibitor (H–Pro–Tyr–(PO3H2)–Leu–Lys–Thr–Lys–Ala–Ala–Val–Leu–Leu–Pro–Val–Leu–Leu–Ala–Ala–Pro–OH) (50 or 250 μmol/L) (Calbiochem, Darmstadt, Germany) for 30 min prior to treatment with leptin (10 μg/mL) for 24 h. To investigate whether leptin-induced IL-1β release is dependent on caspase 1, cells were exposed with BOC-D-FMK (100 μmol/L) (Merck Biosciences, Nottingham, UK) or with Ac-YVAD-CHO (10 μmol/L) (Merck Biosciences) for 30 min prior to treatment with leptin. Cultures were also treated with LPS alone (0.1 μg/mL) (Sigma) for 24 h, or with ATP (5 mmol/L) (Sigma, Poole, UK) in the absence or presence of Ac-YVAD-CHO (10 μmol/L) for 2 h.

ELISA

Interleukin-1β and IL-1RA release was assayed by specific rat sandwich ELISAs, generously provided by Dr Stephen Poole (NIBSC), using specific sheep anti-rat IL-1β or IL-1RA coating antibodies, and specific sheep anti-rat IL-1β or IL-1RA biotinylated antibodies. Recombinant rat IL-1β or IL-1RA was used as standards. The assays were specific for rat IL-1β and IL-1RA with no cross-reactivity with other cytokines. Data were presented as absolute pg/mL values over basal expression. The detection limit of the assay was 20 pg/mL for IL-1β and 19 pg/mL for IL-1RA. The coefficients of variation were 0.5% and 2.1% (intra-assays), and 21.9% and 8.0% (inter-assays), for IL-1β and IL-RA respectively.

Western blot

Western blot analysis for IL-1β, IL-1RA and STAT3 activation was carried out as previously described (Brough et al. 2002), using primary antibodies diluted in phosphate buffered saline containing 0.1% Tween and 0.1% BSA :  sheep anti-rat IL-1β (S328, NIBSC, UK) (1 : 1000 dilution), sheep anti-rat IL-1RA (S377; NIBSC) (1 : 1000 dilution), rabbit anti-STAT3 (New England Biolabs, Hitchin, UK) (1 : 10000 dilution), rabbit anti-phospho-STAT3 (New England Biolabs) (1 : 500 dilution), and subsequent incubation with horseradish peroxidase-conjugated anti-rabbit or anti-sheep IgG secondary antibodies (Dako, Ely, UK) (1 : 2000 dilution).

Data analysis

Results are the mean values ± SEM from 3 to 4 experiments carried out on separate cultures and were analysed using a one-way anova, followed by a Tukey–Kramer post hoc test. For all statistical analyses, a value of p < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Primary rat microglia express leptin receptors mRNA and protein

RT-PCR experiments showed that microglia express mRNAs for the short-isoform (Ob-Ra) and the long-isoform (Ob-Rb) of leptin receptor (Fig. 1A). Ob-Ra and Ob-Rb mRNAs were also detected in mixed glia, astrocytes and neurones. Interestingly, Ob-Rb mRNA expression was much higher in microglia than astrocytes and neurones, whereas Ob-Ra mRNA expression was high in mixed glia, astrocytes and microglia compared with neurones. Ob-R immunostaining was then carried out on microglial cultures using an antibody raised against the C-terminus region of the mouse Ob-R known to recognize both the short- and long-form of the leptin receptor in rat cells (Diano et al. 1998; Hakansson et al. 1998). Strong immunostaining was detected on GSL-IB4 positive microglial cells, with the majority of staining found to be largely localized on the cell surface (Fig. 1B-a,b). The specificity of the immunostaining was confirmed by pre-absorbing the primary antibody with a corresponding blocking peptide (Figs 1B-c).

image

Figure 1.  Expression of leptin receptors. (A) mRNA expression of the short (Ob-Ra) and long (Ob-Rb) isoforms of leptin receptors in mixed glia, microglia, neurones or astrocytes, compared with expression of β-actin mRNA. Single experiment representative of three separate experiments. (B) Ob-R immunostaining in rat microglial cultures using a specific antibody that recognizes both short- and long-isoforms of Ob-R. (a) Lectin (GSL)-IB4 staining. (b) Ob-R immunostaining. (c) Neutralization of Ob-R immunostaining in presence of 10× excess specific blocking peptide. Bar scale = 20 μm

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Leptin induces the release of IL-1β and IL-1RA from microglia

Basal levels of IL-1β and IL-1RA in the medium of untreated microglial cultures were 0.2 ± 0.4 pg/mL and 1020 ± 44 pg/mL respectively. Treatment with recombinant mouse leptin induced a dose-dependent and statistically significant release of IL-1β (Fig. 2a) and IL-1RA (Fig. 2b) over basal levels. Responses to the maximal concentration of leptin (10 μg/mL) were completely abrogated by heat-treatment (95°C for 30 min), suggesting that the effects observed were not due to the presence of endotoxin contamination. Treatment of cells with LPS used as a positive control induced strong release of both IL-1β and IL-1RA.

image

Figure 2.  Effect of treatment with recombinant rat leptin (0.01, 0.1, 0.5, 1, 5, 10 μg/mL), LPS (0.1 μg/mL) or heat treated leptin (HT) (95°C, 30 min) for 24 h on the production of IL-1β or IL-1RA from rat microglial cultures. Data presented are the mean ± SEM of released levels over basal values from four independent experiments carried out on separate cultures. *p < 0.05 and ***p < 0.001 versus control (C), ###p < 0.001 versus leptin (5 and 10 μg/mL). Dotted line represents the detection limit of the assay for IL-β ELISA.

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Leptin-induced release of IL-1β is mediated by activation of the STAT3 signalling pathway

The concentration of leptin (10 μg/mL) that maximally stimulated IL-1β and IL-1RA release from microglia was used to investigate the activation of STAT3, a major signalling component of the leptin signal transduction pathway. STAT3 activation was low in untreated microglia (Fig. 3a). Leptin induced a time-dependent activation of STAT3, reaching statistical significance after 15 and 30 min of treatment. After 60 min of treatment, the level of STAT3 activation returned to basal levels. IFNγ (100 IU/mL) used as positive control, induced maximal activation of STAT3 in microglia after 30 min of treatment. Addition of a specific STAT3 inhibitor peptide (50 or 250 μmol/L) 30 min prior to treatment with leptin significantly reduced leptin-induced IL-1β release, while the inhibitor alone had no effect (Fig. 3b).

image

Figure 3.  Leptin-induced IL-1β release from microglia is dependent on STAT3 activation. (a) Microglial cultures were treated with leptin (10 μg/mL) for 5, 15, 30 or 60 min, or with IFNγ (100 IU/mL) for 30 min, and cell lysates were assayed for STAT3 activation by semi-quantitative western blot analysis. Data presented are the mean ± SEM of three independent experiments carried out on separate cultures. *p < 0.05, **p < 0.01 versus control (C). (b) Microglial cells were treated with specific STAT3 inhibitor peptides (H–Pro–Tyr–(PO3H2)–Leu–Lys–Thr–Lys–Ala–Ala–Val–Leu–Leu–Pro–Val–Leu–Leu–Ala–Ala–Pro–OH) (50 or 250 μmol/L) for 30 min prior to treatment with leptin (10 μg/mL) for 24 h, and IL-1β release was assessed by specific ELISA. Data presented are the mean ± SEM of three independent experiments carried out on separate cultures. ***p < 0.001 versus control (C), ##p < 0.01 versus leptin.

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Leptin induces the release of mature IL-1β by a caspase 1-independent mechanism

In order to determine whether IL-1β release from leptin-treated microglia is dependent on caspases activation (including caspase 1), cells were pre-treated with BOC-D-FMK, a broad spectrum irreversible pan-caspase inhibitor, for 30 min followed by treatment with leptin for 24 h. Leptin-induced IL-1β release was not blocked by BOC-D-FMK, while BOC-D-FMK alone had no effect (Fig. 4a). Similar results were obtained using another caspase inhibitor, Ac-YVAD-CHO, which failed to inhibit leptin-induced IL-1β release but significantly reduced release of IL-1β triggered by ATP from LPS-primed microglia (Fig. 4c). Western blot analysis of the cell lysates showed that leptin induced the synthesis of pro-IL-1β in the cells although at very low-level compared with that induced by LPS (Fig. 4b). Leptin induced the release of pro-IL-1β and mature IL-1β from microglia, which were released at high-level in LPS-treated cells. An additional unidentified IL-1β isoform of 14 kDa was also detected in the culture medium of leptin-treated cells. In addition to IL-1β, leptin induced the synthesis of the 21 kDa precursor isoform of soluble IL-1RA (pro-sIL-1RA) and the release of the 17 kDa soluble mature IL-1RA (Fig. 4b). A similar effect was observed in response to treatment with LPS which induced stronger expression of pro-sIL-1RA and the release of mature sIL-1RA from microglia. BOC-D-FMK had no effect on the cytosolic expression of pro-IL-1β or pro-sIL-1RA, and had no effect on the level of mature IL-1β and sIL-1RA in the medium of leptin-treated cells (Fig. 4b).

image

Figure 4.  Effect of BOC-D-FMK and Ac-YVAD-CHO on leptin-induced IL-1β or IL-1RA synthesis and release in microglial cultures. Cells were treated with BOC-D-FMK (100 μmol/L) or Ac-YVAD-CHO (10 μmol/L) 30 min prior to treatment with leptin (10 μg/mL), LPS alone (0.1 μg/mL) for 24 h, or with ATP (5 mmol/L) in the absence or presence of Ac-YVAD-CHO (10 μmol/L) for 2 h. After 24 h, IL-1β released in the medium was assayed for IL-1β by ELISA (a) (c), and the amount of pro- and mature- IL-1β or IL-1RA was assessed in the cell lysates and the medium by western blot analysis (b). For (a) and (c) data presented are the mean ± SEM of three independent experiments carried out on separate cultures. ***p < 0.001 versus control, ##p < 0.01 versus LPS, §§p < 0.01 versus LPS + ATP. For (b) the results presented are representative of three independent experiments.

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ATP induces the release of 17 kDa mature IL-1β in LPS-primed but not leptin-primed microglia

ATP is a well-known activator of caspase 1 via binding to the P2X7 receptor, resulting in the processing and release of mature IL-1β from LPS-treated microglia (Brough et al. 2002). In order to determine whether ATP can induce the cleavage and release of IL-1β from leptin-treated or LPS-primed cells, microglia were pre-treated with leptin (10 μg/mL) or LPS (0.1 μg/mL) for 24 h, and then treated with ATP (5 mmol/L) for 2 h. Western blot analysis showed that LPS induced strong synthesis of pro-IL-1β in the cells, and the release of some pro- and mature IL-1β in the culture medium (Fig. 5), as already shown in Fig. 4. ATP strongly induced the release of mature IL-1β from LPS-treated microglia, confirming the effect of ATP on IL-1β maturation and release (Brough et al. 2002). ATP also induced the release of pro-IL-1β in the culture medium of LPS-treated cells. In contrast ATP had no effect on the levels of pro- or mature IL-1β detected in the supernatant or cell lysate of leptin-treated microglia.

image

Figure 5.  Effect of ATP on leptin-induced or LPS-induced synthesis and release of IL-1β from microglial cultures. Cells were treated with leptin (10 μg/mL) or LPS (0.1 μg/mL) for 24 h and then treated with ATP (5 mmol/L) for 2 h. Culture medium and cell lysates were collected and analysed for pro- and mature- IL-1β expression by western blot analysis. The results are representative of three independent experiments carried out on separate cultures.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Earlier observations have demonstrated an important role of brain IL-1β in the anorexic, febrile and thermogenic effects of leptin (Luheshi et al. 1999; Sachot et al. 2004; Wisse et al. 2004; Garcia et al. 2006; Harden et al. 2006). The present study shows that microglial cells highly express both isoforms of leptin receptors, Ob-Ra and Ob-Rb, compared with astrocytes or neurones (Fig. 1), and demonstrate that leptin can activate microglia to produce mature IL-1β (Figs 2a and 4). These results add considerably to our understanding of the cellular actions of leptin in the CNS and suggest that the control of food intake and body weight by circulating leptin occurs at least partly via the production of IL-1β from microglial cells of the hypothalamus.

In contrast to undetectable amounts of IL-1β in the medium of untreated microglia, we detected considerable concentrations of its receptor antagonist IL-1RA (Fig. 2b). This observation is in agreement with published studies showing that IL-1RA is readily detectable in both the circulation and brain of normal human and rodents (Palin et al. 2004; Somm et al. 2005), as well as culture medium of untreated primary human liver cells (Gabay et al. 1997). However, one cannot exclude the possibility that microglia in primary cultures are partially activated resulting in high-levels of IL-1RA production. The treatment of microglia with leptin further induced the synthesis and release of IL-1RA over basal levels (Fig. 2b), demonstrating that leptin regulates the production of IL-1RA in parallel with that of IL-1β in microglia. The present in vitro data are in agreement with previous in vivo reports by Hosoi et al. (Hosoi et al. 2002a,b) demonstrating that systemic leptin injection increased mRNA expression of both IL-1β and IL-1RA. Because the balance between IL-1 and IL-1RA will determine the net IL-1 signalling, further investigation is required to characterize the precise role of leptin over the IL-1/IL-1RA ratio and its physiological significance. A recent study showed that serum IL-1RA concentrations are several fold higher in obese individuals, and they decreased after weight-loss (Meier et al. 2002), suggesting a possible implication of IL-1RA in the mechanism of leptin resistance. A separate study revealed that white adipose tissue constitutes a major source of IL-1RA under physiological conditions, and that IL-1RA production increases in obesity as well as in response to inflammatory stimuli (Juge-Aubry et al. 2003). These data combined with the fact that IL-1RA deficient mice show decreased fat mass and are resistant to high-fat diet-induced obesity (Somm et al. 2005), implicate IL-1RA in energy balance regulation.

Although leptin induces the expression and release of IL-1β from microglia, we found that the levels of IL-1β produced were relatively low when compared with those triggered by LPS, which is known to induce robust microglial activation. Whilst it is possible that a higher dose of leptin could induce an amount of IL-1β similar to that induced by LPS, it is plausible that the difference in the levels of this cytokine reflects an alternative physiological role for microglia that is distinct from that normally associated with inflammatory responses to exogenous pathogens or injury. CNS injuries, for example, trigger microglial activation leading to the production of large (pathophysiological) amounts of IL-1 (Allan and Pinteaux 2003). In contrast, leptin may trigger (or maintain) mild microglial activation, which could result in the production of smaller amounts of IL-1β. This low-concentration of IL-1β could contribute to the maintenance of normal body weight as demonstrated by a recent in vivo study by Garcia et al. (2006) using IL-1R1 knockout mice. Furthermore, hypothalamic expression of IL-1β is significantly reduced in response to decreased levels of leptin induced by acute starvation, or in Zucker rats, characterized by dysfunctional leptin receptors (Wisse et al. 2004). These data indicate a role for IL-1β as one of the downstream signals of leptin under physiological conditions.

The hypothesis that microglia and IL-1β may play a physiological role in body weight homeostasis under the influence of leptin, however, does not rule out inflammatory actions of leptin in the brain under pathophysiological conditions. We have shown previously in vivo that administration of leptin in rats induces fever (Luheshi et al. 1999), and that neutralization of endogenous leptin with anti-leptin antiserum attenuates LPS-induced fever (Sachot et al. 2004). More recently, we also reported that leptin induces cyclooxygenase-2 in the brain partly via IL-1 action (Inoue et al. 2006). Interestingly, Sanna et al. (2003) have reported that, in mice autoimmune encephalomyelitis, infiltrating T cells and macrophages produce leptin within the brain, suggesting a role of leptin for the development of certain neuroinflammatory diseases. Although some observations made in the current study would support this notion, further investigations are required to clarify the exact mechanisms that regulate the physiological and pathophysiological actions of this hormone.

Leptin regulates body weight through the activation of STAT3 (Bates et al. 2003), an intracellular signalling molecule activated by Ob-Rb. In the present study, we show that leptin activates STAT3, and demonstrate that inhibition of STAT3 significantly suppressed the leptin-induced IL-1β release in microglial culture (Fig. 3). This finding is somewhat contradictory to previous observations in vivo showing that leptin increased IL-1β mRNA expression in the hypothalamus of obese db/db mice which lack Ob-Rb/STAT3 signalling (Hosoi et al. 2002b). The reason for this discrepancy is unclear, but the different experimental conditions (mRNA levels in mouse brain tissue versus protein levels in microglial culture from rats) can be one possible explanation.

Biological activation of the IL-1β protein depends on proteolytic cleavage of the inactive 32 kDa precursor into a 17 kDa mature form, a mechanism mediated by caspase 1 (Kuida et al. 1995; Li et al. 1995). In the present study, we found that leptin-induced IL-1β release was not inhibited by a broad spectrum pan-caspase inhibitor (BOC-D-FMK) (Fig. 4a and b). Similar results were obtained using a different caspase inhibitor (Ac-YVAD-CHO), which effectively inhibited ATP-induced release of IL-1β from LPS-primed cells (Fig. 4c), a prototypical IL-1β release mechanism mediated by caspase 1. In contrast to its effect on the release of mature IL-1β from LPS-primed cells, ATP failed to induce the release of mature IL-1β from leptin-treated microglia (Fig. 5). These results collectively indicate that leptin-induced IL-1β release occurs independently of caspase 1 activity, and thus involves different mechanisms from LPS-induced IL-1β release. caspase 1-independent processing of IL-1β has already been reported (Miwa et al. 1998) and other extracellular proteases have been proposed for an alternative mechanism of IL-1β cleavage (Schonbeck et al. 1998; Herzog et al. 2005). However, whether or not these mechanisms are involved in the case of leptin was not addressed in the current study. Processing of 32 kDa pro-IL-1β by caspase 1 is thought to lead to the production of a 27 kDa intermediate form, which allows exposure of the cleavage site at Asp116 rendering it accessible to caspase 1 for full processing to the 17 kDa isoform. Western blot analysis showed the presence of an additional band of 14 kDa in the medium of leptin treated cells. An additional 14 kDa isoform of IL-1β has already been reported (Knudsen et al. 1986) and could be the product from direct cleavage of the 32 to the 17 kDa isoform.

Although our data clearly demonstrate that leptin interacts directly with microglial cells in vitro, how this interaction might occur in vivo is still an open question. Given the wide distribution of systemically injected radiolabelled leptin in the brain (Banks et al. 1996) and the abundant distribution of microglial cells throughout the CNS, it is feasible that once in the brain this hormone will activate microglia, regardless of location, resulting in IL-1β production (Hosoi et al. 2002b). However, it is also likely that the action of leptin on microglia occurs more neuroanatomically restricted to areas of the brain (for example the hypothalamus) as a result of a more restricted entry to specific regions such as those described for astrocytes acting as a delivery system for leptin to the arcuate nucleus of the hypothalamus (Cheunsuang and Morris 2005).

In summary, these observations suggest that microglia are a target for leptin action which leads to the production of the pro-inflammatory cytokine IL-1β. In combination with our previous findings in vivo (Luheshi et al. 1999), these results add further support to the hypothesis that leptin acts as a neuroimmune modulator, and suggest that microglial cells play an important part in this process.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by the Medical Research Council UK, the Canadian Institutes of Health Research and Natural Sciences and Engineering Research Council of Canada. The authors would like to thank Drs Adrian Bristow and Stephen Poole (NIBSC, UK) for providing recombinant mouse leptin and rat specific ELISAs.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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