Published online before print July 7, 2004
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,1
* Charité Universitaetsmedizin Berlin, Campus Benjamin Franklin, Germany;
Department of Medicine, University of Colorado Health Sciences Center, Denver; and
Institute of Pathology, University of Mainz, Germany
1Correspondence: University of Colorado, Department of Medicine, Box B168, 4200 East Ninth Ave., Denver, CO 80262. E-mail: Giamila.Fantuzzi{at}uchsc.edu
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, IL-6, and IL-13 from colon cultures and splenocytes did not differ among these two groups. Conversely, rates of apoptosis were higher in lamina propria lymphocytes obtained from the colon of IL-10 KO ob/ob compared with IL-10 KO mice. In conclusion, although leptin deficiency has been associated with resistance in models of autoimmunity and inflammation induced by exogenous stimuli, leptin appears not to play a significant role in the spontaneous colitis of IL-10 KO mice, although it modulates survival of intestinal lymphocytes.
Key Words: obesity • cytokines • colon • leptin
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Leptin deficiency—in humans and mice—is associated with reduced production of several cytokines [3
4
5
]. The pattern of cytokines regulated by leptin seems to differ with the type of stimulus used. For example, leptin-deficient mice produce low levels of tumor necrosis factor 
(TNF-) and interleukin (IL)-18 but not interferon- (IFN-) or IL-12, following administration of concanavalin A (Con A), whereas in the same mice, injection of endotoxin leads to production of low levels of IL-10 and IL-1 receptor antagonist but not TNF- or IL-1ß [6
, 7
].
Mice deficient for production of leptin (ob/ob mice) are obese and resistant in a variety of experimental models of inflammation or autoimmunity induced by administration of exogenous substances [5 , 8 , 9 ]. In particular, ob/ob mice are resistant to intestinal inflammation induced by dextran sulfate sodium or trinitrobenzene sulfonic acid to experimental autoimmune encephalomyelitis induced by immunization with myelin basic protein peptides and to arthritis induced by administration of methylated bovine serum albumin [8 9 10 ]. In these models, resistance to disease in the absence of leptin is associated with reduced cytokine production and in the two latter models, with a shift from a T helper cell type 1 (Th1) to a Th2 pattern of cytokine responses. In humans, a possible role for leptin in regulating intestinal inflammation is suggested by data indicating an increased expression of leptin mRNA in the mesenteric adipose tissue obtained from patients with inflammatory bowel disease [11 ]
Despite these important observations, the role of leptin in regulating inflammatory or autoimmune responses developing spontaneously as a result of alterations in the immune system—a situation that more closely reflects human autoimmune disease—has not been studied in great detail. The only published study about this topic demonstrates that in the nonobese diabetic (NOD) model of type I diabetes, administration of exogenous leptin accelerates destruction of insulin-producing ß cells and increases production of IFN- [12
].
IL-10-deficient [IL-10 knockout (KO)] mice spontaneously develop chronic intestinal inflammation, which is mediated by CD4+ T cells and is associated with enhanced Th1 responses in the early course of disease, and Th2 cytokines progressively increase later on [13 , 14 ]. As colitis in IL-10 KO mice is dependent on the very cell types and cytokines that are thought to be regulated by leptin, we generated leptin-deficient IL-10 KO mice (IL-10 KO ob/ob) to evaluate the role of leptin in a model of spontaneously developing inflammation. Our results indicate that leptin does not play an important role in modulating inflammation in the IL-10 KO model.
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Clinical and histological assessment of colitis
Mice were weighed weekly and monitored for appearance of diarrhea and blood in the stools. A disease scoring system was used as follows: Appearance of diarrhea was scored as 0 = well-formed pellets, 2 = pasty and semi-formed stools that did not adhere to the anus, 4 = liquid stools that did adhere to the anus. Appearance of blood in the stools was scored as 0 = no blood using hemoccult (Beckman Coulter, Palo Alto, CA), 2 = positive hemoccult, 4 = gross bleeding. Development of rectal prolapse was scored as 0 = no prolapse, 2 = prolapse evident only during defecation, 4 = prolapse evident at all times. The total scores given for diarrhea, stool blood, and prolapse were added and divided by 3, for a maximal disease score of 4. Postmortem, the entire colon was excised, and a 1-cm segment of the transverse colon was fixed in 10% buffered formalin for histological analysis. Paraffin sections were stained with hematoxylin/eosin. Four to six colon rings were obtained from each 1 cm colon segment and were thus available for histological examination. Histological scoring was performed in a blinded manner by a pathologist (H. A. Lehr) as a combined score of inflammatory cell infiltration (0–3) and tissue damage (0–3) as described [16
].
Colon culture
A segment of the colon was removed, cut open longitudinally, and washed in phosphate-buffered saline containing penicillin and streptomycin. The colon was then cut into strips of 
1 cm2 and placed in 24 flat-bottom well culture plates containing 1 ml RPMI supplemented with penicillin and streptomycin. Strips were incubated at 37°C for 24 h. Culture supernatants were then harvested and assayed for cytokines.
Preparation of intraepithelial (IEL) and lamina propria (LPL) cells
IEL and LPL were isolated from the small intestine and the colon as described previously [10
].
Preparation and culture of spleen cells
Single-cell suspensions of splenocytes were prepared by passing cells through a 100-µM strainer. Splenocytes were cultured for 24 h at 2.5 x 106/ml in 96-well plates in the presence of Con A (1 µg/ml) or plate-bound anti-CD3
antibodies (BD PharMingen, San Diego, CA) at 1 µg/ml with or without soluble anti-CD28 antibodies at 1 µg/ml.
Cytokine measurements
IFN-, IL-6, and IL-13 were measured using specific enzyme-linked immunosorbent assay (BD PharMingen and R&D Systems, Minneapolis, MN).
Flow cytometry
Flow cytometry followed routine procedures using 1 x 105 cells per sample. Staining with Annexin V and propidium iodide (PI; BD PharMingen) was used for evaluation of the rate of apoptosis in LPL. Surface-marker expression was evaluated using antibodies from BD PharMingen and Caltag (S. San Francisco, CA). Analysis was conducted on a FACSCalibur (BD PharMingen) using the CellQuest analysis program (BD PharMingen).
Statistical analysis
Data are expressed as mean ± SEM. Statistical significance of differences between treatment and control groups was determined by factorial ANOVA. Statistical analyses were performed using the XLStat software (Addinsoft, Brooklyn, NY).
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Figure 1. Body weight (BW) in IL-10 KO and IL-10 KO ob/ob mice. WT (n=16), IL-10 KO (n=16), ob/ob (n=10), and IL-10 KO ob/ob (n=9) mice were weighed weekly starting at an age of 4 weeks. P < 0.001 for WT versus IL-10 KO and ob/ob versus IL-10 KO ob/ob at all time-points; P < 0.0001 for WT and IL-10 KO versus ob/ob or IL-10 KO ob/ob at all time-points. Data are mean ± SEM.
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Figure 2. Clinical disease and histologic score in IL-10 KO and IL-10 KO ob/ob mice. (A) Clinical disease score was calculated weekly for IL-10 KO (n=16) and IL-10 KO ob/ob (n=12) mice. Differences between the two groups did not reach statistical significance. (B) Histologic score was calculated at week 16. Data are mean ± SEM. ***, P < 0.001, versus WT or ob/ob mice. Differences between IL-10 KO and IL-10 KO ob/ob mice did not reach statistical significance.
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and IL-6 was evaluated from unstimulated colon cultures in IL-10 KO and IL-10 KO ob/ob mice at 16 weeks of age. As shown in Figure 3
, no significant difference in production of these two cytokines was observed. Furthermore, splenocytes obtained from IL-10 KO and IL-10 KO ob/ob mice were stimulated with anti-CD3 and anti-CD28, and production of IFN-, IL-6, and IL-13 was evaluated. Again, no significant difference was observed for production of any of these cytokines (Table 1 ). Similarly, LPL obtained from the colon of IL-10 KO and IL-10 KO ob/ob mice produced similar levels of IFN- when stimulated with Con A or plate-bound anti-CD3 (Table 2
).
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Figure 3. Cytokine production from colon cultures. Colon cultures of tissue obtained at week 16 from WT, ob/ob, IL-10 KO, and IL-10 KO ob/ob mice were performed. Levels of IFN- and IL-6 were measured in the supernatant. Data are mean ± SEM of five mice per group. *, P < 0.05; ***, P < 0.001, versus WT or ob/ob mice. Differences between IL-10 KO and IL-10 KO ob/ob mice did not reach statistical significance.
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Table 1. Cytokine Production in Splenocytes Obtained from IL-10 KO and IL-10 KO ob/ob Mice
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Table 2. IFN- Production in LPL Obtained from IL-10 KO and IL-10 KO ob/ob Mice
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ß, TCR
, CD4, CD8, and CD8ß did not differ in IEL and LPL obtained from the colon or the small intestine of IL-10 KO and IL-10 KO ob/ob mice (data not shown), although a trend toward a lower percentage of double-positive CD4 CD8 cells was observed in IEL and LPL populations. However, consistent with a role for leptin in modulating T cell death, a higher rate of apoptotic LPL was present in the colon of IL-10 KO ob/ob mice compared with IL-10 KO mice (Fig. 4
).
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Figure 4. High rates of apoptosis in LPL obtained from IL-10 KO ob/ob mice. Colonic LPL were isolated at week 16 from IL-10 KO and IL-10 KO ob/ob mice. Rates of apoptosis were measured by flow cytometry using Annexin V/PI staining. A representative figure obtained from one IL-10 KO and one IL-10 KO ob/ob mouse is shown. A total of three mice per group was evaluated.
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Despite developing severe intestinal inflammation, IL-10 KO ob/ob mice are still markedly obese, suggesting that obesity secondary to leptin deficiency does not require the presence of IL-10. However, similar to IL-10 KO mice, body weight is significantly reduced in IL-10 KO ob/ob mice compared with their littermates not carrying a deletion of the IL-10 gene. As reduced body weight in IL-10 KO mice is a consequence of intestinal inflammation [14
], these data indicate that disease-associated anorexia and body weight loss are not leptin-dependent. These results are in agreement with previous reports indicating that administration of endotoxin, TNF-, or IL-1 can induce anorexia and body weight loss in leptin-deficient and leptin receptor-deficient animals [18
, 19
].
Leptin is considered an important regulator of production of several cytokines, particularly IFN- [3
, 8
9
10
]. Reduced cytokine production in ob/ob mice is generally associated with disease amelioration, making it difficult to dissociate a possible primary effect of leptin on cytokine production from a secondary reduction as a result of development of less severe disease. In the present report, we demonstrate that production of the cytokines IFN-, IL-6, and IL-13 is not altered in IL-10 KO ob/ob mice, demonstrating that the presence of bioactive leptin is not an absolute requirement for these cytokines to be synthesized. These data are in agreement with previous reports demonstrating that the effect of leptin on cytokine production is variable depending on the type of stimulus used [5
, 6
, 10
, 20
].
IL-10 KO ob/ob mice develop intestinal inflammation to a similar degree to that observed in their IL-10 KO littermates in terms of clinical disease score and histologic examination of the colon. This observation is fundamentally different from previous data, indicating that ob/ob mice are protected in models of intestinal inflammation induced by administration of exogenous stimuli [10 , 21 ] and that leptin receptor deficiency on T lymphocytes delays disease development in the model of colitis induced by transfer of CD4+ CD45RBhigh splenocytes into scid recipients [22 ]. Our present data thus contrast with the concept that ob/ob mice are protected in various models of autoimmune disease and inflammatory conditions [5 , 8 9 10 ]. A critical difference between our present report and those previous studies resides in the requirement of exogenous substances or manipulation of cell populations: In IL-10 KO mice, intestinal inflammation develops spontaneously, as the results of "spontaneous" breaking of tolerance toward the enteric flora, whereas each of the alternative models studied to date requires external manipulations in the form of immunization, administration of exogenous substances, or transfer of cells to preactivate the immune system and eventually induce disease. Although similar cell types and soluble mediators are involved in development of autoimmunity and inflammation in each of the above-mentioned models and in the IL-10 KO mouse, our data suggest that fundamentally different mechanisms trigger disease in the two types of systems. In this context, it is important to note that colitis in IL-10 KO mice results from an unopposed immune and inflammatory response to the normal enteric flora [23 ]. Leptin and IL-10 use signal transducer and activator of transcription 3 (STAT-3) as a critical signal-transduction pathway [10 , 24 ]. Although IL-10-induced activation of STAT-3 in macrophages generates anti-inflammatory effects [24 ], the same signaling pathway leads to proinflammatory activities when leptin is the inducer [25 ]. Our results indicate that in the absence of leptin and IL-10, the net effect of the lack of the anti-inflammatory mechanisms and subsequent lack of tolerance to colonic bacetria mediated by IL-10 is predominant and cannot effectively be counter-regulated by the absence of leptin. It should be noted that the data presented herein on the IL-10 KO mouse model of spontaneous colitis may not necessarily be transferred to other models of spontaneous colitis in which the specific balance of inflammatory mediators may well leave a critical role for leptin, as suggested in previous studies in NOD mice [12 ]. Of interest, IL-10 seems to play a pro- rather than antipathogenic role early in disease development in the NOD mouse model of diabetes, indicating a possible inter-relationship between the effects of leptin and IL-10 in regulating immune responses [26 ].
In the clinical situation, a possible role for leptin in participating in the regulation of intestinal inflammation is suggested by studies showing increased leptin expression in mesenteric adipose tissue obtained from patients affected by Crohn’s disease or ulcerative colitis compared with a control population [11 ]. However, as the primary pathogenic cause of inflammatory bowel disease is still largely unknown, only more extensive clinical trials in patients affected by Crohn’s disease or ulcerative colitis will eventually indicate whether and to what extent leptin contributes to intestinal inflammation.
Received April 16, 2004; revised May 27, 2004; accepted June 13, 2004.
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and IL-18 Proc. Natl. Acad. Sci. USA 97,2367-2372
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