INTRODUCTION

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INTERLEUKIN (IL)-1 is one of the key mediators of the host response to tissue injury, infection, or inflammation. Administration of IL-1 itself or of inducers of IL-1, such as lipopolysaccharide (LPS) or turpentine, mimics many of the pathophysiological changes that occur during infection or inflammation, such as anorexia, fever, induction of acute phase proteins, hypoglycemia, and activation of the hypothalamic-pituitary-adrenal (HPA) axis (8, 26).

The biological activity of IL-1 is mediated by two different gene products, IL-1 and IL-1, which share similar biological activities by binding to the same receptors. IL-1 remains mostly membrane associated or within cells, whereas IL-1 is usually secreted (8). With the use of IL-1-deficient (IL-1 /) mice, IL-1 has been shown to be an essential mediator of the inflammatory, pyrogenic, and anorectic response elicited by the local tissue damage caused by subcutaneous injection of turpentine (33). Studies with antibodies to IL-1 have also shown that IL-1 contributes to LPS-induced fever (20). However, the generation of the inflammatory responses to LPS, such as anorexia, activation of HPA axis, increases in IL-1, tumor necrosis factor (TNF)-, and IL-6, and the induction of acute phase proteins, are observed even in absence of IL-1 expression (12).

IL-6 is another mediator of the host response to inflammation. IL-6 / mice do not develop an acute phase response (APR) or anorexia in response to turpentine (13). Moreover, IL-6 has been shown to be the primary mediator of the fever induced by either LPS or IL-1 (3). However, IL-6 / mice exhibit anorexia, hypoglycemia, induction of acute phase proteins, and activation of the HPA axis after LPS administration (13). In the inflammatory response induced by LPS, multiple cytokines with overlapping activities, including IL-1, IL-1, TNF- and IL-6, are simultaneously induced, whereas turpentine specifically induces only IL-1 and IL-6 without any detectable increase in TNF- or IL-1 (10, 11). Therefore, multiple cytokines contribute to the development of the APR elicited by LPS, but it appears that only IL-1 and IL-6 regulate the inflammatory response to turpentine.

Leptin (Ob protein), the product of the ob gene, is a 16-kDa protein synthesized by adipose tissue that plays a crucial role in the homeostasis of body weight by regulating food intake and energy expenditure (32). Administration of leptin to rodents decreases food intake and body weight and increases energy consumption (25). Fasting and starvation decrease leptin mRNA and protein levels, and this substantial fall represents a signal to eat (6, 22). Defects in leptin or leptin receptor gene expression cause severe obesity in rodents (4, 31).

We have shown that administration of LPS, IL-1, or TNF- acutely increases steady-state levels of the mRNA for leptin in adipose tissue and circulating levels of leptin in hamsters (17). In addition, Sarraf and colleagues (28) have demonstrated that LPS and proinflammatory cytokines induce leptin expression in mice. These data suggest that leptin induction during inflammation is regulated in a manner similar to the cytokine response to infection and injury. The observation that the leptin receptor is homologous to the gp130 family, the signal-transducing subunit of the IL-6-type cytokine receptors (4), further supports the hypothesis that leptin might play a role in the inflammatory response. Moreover, recent work showed that leptin stimulates the immune system, because leptin enhances cytokine production and phagocytosis by macrophages (16). A direct effect of leptin on development and differentation of hemopoietic lineages has also been described (23).

To further clarify the regulation of leptin induction during the host response to infection and inflammation, we determined whether other stimuli, such as local sterile tissue damage induced by the subcutaneous injection of turpentine, can induce leptin mRNA expression and leptin synthesis in mice. In addition, using IL-1 / and IL-6 / mice, we determined the specific role played by these cytokines in leptin induction after turpentine and LPS administration.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results & Discussion References

Materials. [³²P]dCTP (3,000 Ci/mmol) was purchased from NEN (Boston, MA), LPS (a phenol-extracted preparation from E. coli serotype O55:B5) was from Sigma (St. Louis, MO), multiprime DNA labeling system kits were purchased from Amersham Life Sciences International (Arlington Heights, IL), Minispin Sephadex G-50 columns were from Worthington Biochemical (Freehold, NJ), and Nytran+ nylon membranes were from Schleicher and Schuell (Keene, NH).

Animals and treatments. IL-1 / mice were generated as previously described (33). IL-6 / mice were purchased from Jackson Laboratory, Bar Harbor, ME. Four- to six-week-old male IL-1 +/+ and IL-1 / or IL-6 +/+ and IL-6 / mice of mixed C57BL/6 and 129/Sv background, housed five per cage, were used. LPS was administered intraperitoneally at a dose of 5 mg/kg, a dose of LPS that has been previously shown to induce APR in mice (12). Steam-distilled turpentine was injected subcutaneously at a dose of 100 µl in the hindlimb. Control mice were injected intraperitoneally or subcutaneously with sterile, pyrogen-free saline. All animal protocols were approved by the Animal Studies Committee of the San Francisco Veterans Affairs Medical Center.

Isolation of RNA and Northern blotting. Total RNA was isolated from epididymal adipose tissue by modification of the method of Chomczynsky and Sacchi (5). Fat tissue obtained from each mouse was individually processed. Lipid extraction with CHCl₃ was performed immediately after homogenization; the aqueous phase was then acidified and subjected to the standard acid-phenol-chloroform extraction. Gel electrophoresis, transfer, Northern blotting, and densitometry were performed as previously described (14). A murine ob gene cDNA probe, generated as previously described (17), was used. Because we and others have shown that LPS and cytokines increase mRNA levels of "housekeeping" genes such as actin and cyclophilin, two mRNAs commonly used for normalizing data, we loaded equal amounts of total RNA (10 µg, determined by spectrophotometry) and assessed uniformity of sample application by ultraviolet visualization of the acridine orange-stained ribosomal RNAs in the gel before electrophoretic transfer. We have previously reported that the effects of LPS or cytokines are specific for individual mRNAs in individual tissues both in terms of increased or decreased levels and of the order of magnitude of the change. For example, treatment of hamsters with LPS or cytokines decreases the level of cholesterol ester transfer protein mRNA in adipose tissue (18). In liver, specific mRNAs may be either increased or decreased to varying degrees ranging from 30-fold increases to 95% decreases (14, 15).

Leptin and IL-6 measurement. Serum leptin levels were measured using a radioimmunoassay kit specific for mouse leptin (Linco Research, St. Charles, MO). Serum IL-6 levels were measured using a mouse enzyme-linked immunosorbent assay kit, kindly provided by Endogen (Cambridge, MA).

Statistical analysis. Analysis of variance with post hoc Bonferroni's test was used. Data are expressed as means ± SE.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials & Methods Results & Discussion References

To explore whether inflammatory stimuli other than LPS were able to induce leptin, we tested the effect of subcutaneous turpentine (100 µl) on leptin levels in IL-1 +/+ and IL-1 / mice. Control mice received subcutaneous saline. As shown in Fig. 1, A and B, high levels of leptin in serum and leptin mRNA in adipose tissue were detected in fed IL-1 +/+ and IL-1 / mice compared with fasted mice. However, despite fasting, which normally suppresses leptin expression (1), turpentine markedly induced leptin synthesis and mRNA levels 16 h after treatment in IL-1 +/+ mice. Time course experiments demonstrated that 16 h was the time of maximal induction of leptin (data not shown). In contrast, in IL-1 / mice, the induction of leptin gene expression and synthesis after turpentine was dramatically impaired, demonstrating that IL-1 is essential for turpentine-induced leptin.

View larger version (21K): [in this window] [in a new window]   View larger version (40K): [in this window] [in a new window]   Fig. 1.   Turpentine increases leptin serum and adipose tissue mRNA levels in interleukin (IL)-1 +/+ but not in IL-1-deficient (/) mice. IL-1 +/+ and IL-1 / mice were injected with turpentine (100 µl sc) and then fasted. Saline-treated mice were fasted or fed ad libitum. Blood and epididymal tissue were removed 16 h after treatment, and leptin serum (A) and mRNA levels (B) were measured as described in MATERIALS AND METHODS. Values are means ± SE; n = 5 for each group. * P < 0.05; *** P < 0.001 vs. IL-1 +/+ mice treated with saline and then fasted, by unpaired measures analysis of variance (ANOVA) with post hoc Bonferroni's test.

To elucidate the role of IL-1 in leptin production in another model of inflammation, IL-1 +/+ and IL-1 / mice were challenged with LPS (5 mg/kg ip) and then fasted. Control fasted mice were injected intraperitoneally with saline. Despite fasting, which under physiological conditions is accompanied by low levels of leptin, leptin protein (Fig. 2A) and mRNA levels (Fig. 2B) were markedly increased 6 h after LPS treatment in IL-1 +/+ mice. Time course experiments showed that LPS induced maximal expression of leptin at 6 h (data not shown). When LPS was injected in IL-1 / mice, however, leptin synthesis and mRNA levels did not increase over basal levels, demonstrating that IL-1 also mediates the leptin response to LPS, a model of systemic inflammation.

View larger version (17K): [in this window] [in a new window]   View larger version (31K): [in this window] [in a new window]   Fig. 2.   Lipopolysaccharide (LPS) increases leptin serum and adipose tissue mRNA levels in IL-1 +/+ but not in IL-1 / mice. IL-1 +/+ and IL-1 / mice were injected with LPS (5 mg/kg ip) or saline and then fasted. Blood and epididymal tissue were removed 6 h after treatment, and leptin serum (A) and mRNA levels (B) were measured as described in MATERIALS AND METHODS. Values are means ± SE; n = 5 for each group. ** P < 0.01; *** P < 0.001 vs. IL-1 +/+ mice treated with saline and then fasted, by unpaired measures ANOVA with post hoc Bonferroni's test.

The requirement for IL-1 in the induction of leptin during turpentine-induced inflammation is consistent with the essential role played by IL-1 in turpentine-induced APR (33). However, the result that IL-1 was also essential for LPS induction of leptin was unexpected. After turpentine, IL-1 and IL-6 are the sole detectable cytokines, and they are required for the development of the systemic APR. In fact, no APR is observed after turpentine in either IL-1 / or IL-6 / mice (13, 33). In contrast, the systemic response to LPS is characterized by the induction of several cytokines, such as TNF, IL-1, and IL-6 (10). These cytokines have pleiotropic and redundant activities and exhibit a pattern of mutual induction, modulating each other's production. Moreover, each of these cytokines is independently induced by LPS; therefore no single cytokine is considered crucial for most LPS-induced responses. However, despite the redundancy that characterizes many of the responses to LPS, the induction of leptin after LPS appears to be completely under the control of IL-1.

As shown in Table 1, both LPS and turpentine increased serum IL-6 in IL-1 +/+ mice. IL-6 serum levels were measured at the time of maximal induction for each agent, 3 h for LPS and 16 h for turpentine. As previously shown (12, 33), the induction of IL-6 after LPS administration was only slightly reduced in IL-1 / mice, whereas turpentine did not induce IL-6 in IL-1 / mice. Therefore, either LPS directly induces IL-6, or other cytokines with overlapping activities appear to compensate for the absence of IL-1 for the production of IL-6 after LPS. In contrast, IL-1 is essential for IL-6 production during turpentine-induced inflammation, suggesting a cascade in which turpentine first induces IL-1, which in turn induces IL-6. Moreover, IL-6 / mice do not develop an APR in response to turpentine (13, 19). Therefore, the impaired inflammatory response to turpentine observed in IL-1 / mice appears to be a consequence of the absence of the induction of IL-6, which mediates the APR initiated by turpentine.

View this table: [in this window] [in a new window]   Table 1.   Serum IL-6 levels after LPS or turpentine in IL-1  +/+ and IL-1  / mice

To evaluate the role of IL-6 in IL-1-mediated induction of leptin after turpentine, we studied the reponse of IL-6 / mice. As shown in Fig. 3, no differences were observed between IL-6 +/+ and IL-6 / mice in circulating levels of leptin 16 h after turpentine. The equivalent response induced in both groups of mice indicates that leptin induction by turpentine occurs even in the absence of IL-6, demonstrating that IL-1 independently induces both IL-6 and leptin (Fig. 4).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Turpentine increases serum leptin both in IL-6 +/+ and IL-6 / mice. IL-6 +/+ and IL-6 / mice were injected with turpentine (100 µl sc) or saline and then fasted. Blood was collected 16 h after treatment, and leptin serum levels were determined. Values are means ± SE; n = 10 for each group. ** P < 0.01 vs. IL-6 +/+ mice treated with saline and then fasted, by unpaired measures ANOVA with post hoc Bonferroni's test.

View larger version (8K): [in this window] [in a new window]   Fig. 4.   Induction of cytokines by turpentine and LPS. Turpentine specifically induces IL-1 production, which then independently stimulates the synthesis of IL-6 and leptin. On the other hand, after LPS injection, several proinflammatory cytokines are induced. However, IL-1 is primarily the inducer of leptin production. TNF-, tumor necrosis factor-.

The increase in leptin after either turpentine or LPS indicates that leptin is part of the host's cytokine response to inflammation. Leptin regulates feeding behavior and therefore may be a mediator of the anorexia associated with chronic and acute inflammation. However, we have shown that ob/ob and db/db mice, which are defective in leptin and leptin receptor, respectively, are responsive to LPS in terms of anorexia (9), suggesting that leptin is not the sole cause of the anorexia induced by LPS. These results are in agreement with the finding that LPS induces anorexia even in absence of IL-1 and therefore in the absence of induction of leptin (12).

The primary role of IL-1 in the induction of leptin during inflammation suggests that some of the biological activities of IL-1 itself may be specifically mediated by leptin. IL-1 is anorexigenic, and leptin might contribute to IL-1-induced anorexia (30). However, it has been shown that central administration of IL-1 induced greater anorexia in fa/fa rats, which have a mutation in the leptin receptor and display the same phenotype as the db/db mice, than in lean rats (27). Thus centrally injected IL-1 might induce anorexia through a different pathway than when injected or produced in the periphery. A selective subdiaphragmatic vagal deafferentation does not block anorexia induced by peripheral LPS or IL-1 (29), supporting the concept that different mechanisms are involved in the anorexia induced by central or peripheral stimuli. It is therefore possible that IL-1-induced leptin in the periphery could be part of the mechanism of the anorexia induced by peripherally injected IL-1.

Leptin regulates energy balance not only by decreasing food intake, but also by increasing energy expenditure via activation of the sympathetic nervous system (25). The pyrogenic and thermogenic response to IL-1 is impaired in fa/fa rats (7). Therefore, leptin may contribute to thermogenesis during inflammation by mediating the increase in brown adipose tissue sympathetic and thermogenic activity induced by IL-1.

On the other hand, in addition to regulating food intake and energy expenditure, leptin may have other physiological functions. Constitutive levels of mRNA for leptin receptor are present not only in the hypothalamus, but also in regions of the brain not directly involved in the regulation of food intake. Moreover, ob/ob and db/db mice are not only obese but also have a variety of hormonal and metabolic disorders, including infertility and a dysfunctional adrenal and thyroid axis. Several reports showed an important role for leptin in reproductive physiology (2). Alterations in thyroid, adrenal, and reproductive functions occur during infection and inflammation, and it is possible that leptin mediates or modulates, at least in part, these neuroendocrine responses.

In addition, a short isoform of leptin receptor mRNA is expressed in many peripheral tissues and cells, including kidney, lung, liver, spleen, and macrophages (23, 31) and recently has been shown to be capable of transmitting signals (24). Moreover, leptin has been shown to enhance cytokine production and phagocytosis by macrophages and stimulates hematopoiesis (16, 23). This potential role in the hemopoietic and immune system development might also explain the impairments seen in the immune system of ob/ob mice.

In summary, our data show that leptin is induced after injection of turpentine or LPS and that IL-1 has an essential role in the induction of leptin during local and systemic inflammation.