Leptin is a small 16-KDa protein produced primarily by

A role for leptin in sustaining lymphopoiesis and myelopoiesis Kate Claycombe*, Louis E. King, and Pamela J. Fraker* Departments of *Food Science and Human Nutrition and Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319 Contributed by Pamela J. Fraker, December 20, 2007 (sent for review December 6, 2007) Although leptin is known for its regulation of food intake, it has many emerging roles in immune function. To better define the role of leptin in hematopoietic processes, a leptin-deficient obese mouse (ob/ob) and C57BL/6 lean wild-type controls were compared. Despite their large size and consumption of substantial amounts of nutrients, the ob/ob mice had only 60% as many nucleated cells in their marrow as controls. The greatest impact of leptin deficiency was on the B cell compartment that had 70% fewer cells, reducing the absolute number of pre-b and immature B cells to 21% and 12% of normal, respectively, and indicating a significant reduction in lymphopoiesis in ob/ob mice. Whereas the proportion of myeloids remained nearly normal in the obese mice, they also exhibited a reduction of 40% and 25%, respectively, in absolute numbers of granulocytes and monocytes. Seven days of provision of recombinant leptin promoted substantial lymphopoiesis, increasing the numbers of B cells in the marrow of the obese mice twofold, while doubling and tripling, respectively, the numbers of pre-b and immature B cells. Twelve days of supplementation brought these subpopulations to near-normal proportions. Leptin treatment also facilitated myelopoiesis such that the marrow of the obese mice contained normal numbers of monocytes and granulocytes after 7 days. Taken together, the data support an important role for leptin in sustaining lymphopoiesis and myelopoiesis. obesity ob/ob mouse pre-b cell pre-t cell Leptin is a small 16-KDa protein produced primarily by adipocytes (1). It acts on regions of the hypothalamus and controls food intake and body temperature (1, 2). The severe degree of obesity found in ob/ob mice that are deficient in functional leptin and db/db mice that lack functional leptin receptors bears witness to the major role leptin plays in regulating energy homeostasis and food intake (3, 4). However, these two strains of mice also manifest defects in innate and adaptive immunity (5, 6). Indeed, the literature demonstrates that leptin can modulate both neuroendocrine (7) and immune functions (6 11). CD34 marrow precursor cells (12), T cells (12), B cells, and phagocytic cells including promyelocytes all bear leptin receptors, indicating potential modulation of their function or metabolism by leptin (7, 13, 14). Interestingly, leptin receptors are members of the class I cytokine receptor family and leptin itself contains four -helix motifs analogous to IL-6 family members (7, 15). Leptin often is present during acute-phase responses that include C-reactive protein, serum amyloid A, and tumor necrosis factor (16, 17). Collectively, this information indicates that leptin modulates a variety of immune functions. Evidence that leptin plays some role in maintaining the integrity of the primary immune tissues has also emerged. However, the literature on the possible roles for leptin in hematopoietic functions is limited by being contradictory, as pointed out in a past review (13). For example, in one study it was suggested that leptin promotes myelopoiesis to the exclusion of effects on other lineages in the marrow (18). However, these studies were performed in vitro in serum-free media. The addition of leptin promoted the formation of only a modest number of granulocyte-macrophage-like colonies. Surprisingly, the addition of G-CSF or GM-CSF did not promote additional myeloid colony formation. Conversely, when others compared colony formation capacity for wild-type and ob/ob mice, they found both produced lymphoid and myeloid colonies, although fewer numbers of colonies were produced by marrow from the obese mice (12). Recent evidence that leptin can promote thymopoiesis has been clearly demonstrated (19). It was found that leptin injection of ob/ob mice that have involuted thymuses promoted robust thymopoiesis (19). Similarly, the leptinreceptor-defective db/db mice also display atrophied thymuses and reductions in thymus cell number, indicating a dependency on leptin (20). We were interested in further exploring the role of leptin in lymphopoiesis and myelopoiesis. The experiments described herein were performed in vivo comparing and contrasting hematopoietic processes in lean wild-type C57BL/6J mice (control) versus their mutated ob/ob facsimile that are deficient in functional leptin. In addition, control and ob/ob mice were injected with recombinant leptin to evaluate its impact on the lymphopoietic and myelopoietic compartments of the leptin-deficient mice. The data will show that leptin appears to play an essential role sustaining lymphopoiesis and myelopoiesis in the marrow. Results Modulation of Body Weight and Food Intake by Leptin. The powerful impact of leptin on metabolism, especially food intake, is shown here (Fig. 1). Control mice injected with either leptin or saline consumed 3.5 to 3.6 g of diet per day per mouse. The untreated ob/ob mice consumed 6.35 g of diet per day per mouse during the same period and gained an additional 4gofbody weight such that their final weight was 2.6 times that of controls (Fig. 2). Conversely, the 7-day regimen of leptin injections reduced ob/ob intake to near-normal amounts, creating a weight loss of 2 g (Fig. 2). Control mice treated with leptin lost 0.5 g of body weight, whereas untreated control mice gained 1 g of weight during the 7-day period. Impact of a Deficiency of Leptin on Lymphopoiesis and Myelopoiesis. The next data provide an overview of the impact of obesity and the accompanying deficiencies in leptin on the major lineages developing in the marrow of the mouse (Fig. 3). By using protocols and gating procedures outlined in earlier publications, anti-cd 31 and anti-ly6c were used to divide the marrow into five major compartments (21, 22). In addition, anti-ly6g (Gr-1), anti-cd45r, and anti-ter119 were used to further verify the regions containing granulocytic cells, cells of the B lineage, and erythroid lineages, respectively. Of immediate interest was the 50% decrease in the proportion of cells in the lymphoid compartment of the ob/ob mice Author contributions: K.J.C., L.E.K., and P.J.F. designed research; K.J.C. and L.E.K. performed research; K.J.C., L.E.K., and P.J.F. analyzed data; and K.J.C. and P.J.F. wrote the paper. The authors declare no conflict of interest. To whom correspondence should be addressed. E-mail: fraker@msu.edu. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0712053105/DC1. 2008 by The National Academy of Sciences of the USA www.pnas.org cgi doi 10.1073 pnas.0712053105 PNAS February 12, 2008 vol. 105 no. 6 2017 2021

Fig. 1. The effect of leptin on food intake in C57BL/6J (control) and ob/ob (leptin-deficient) mice. Food and body weights were measured every day for 7 days. The mean values were calculated by averaging daily food consumption per mouse for all groups. C, control; O, obese; OL, obese with leptin treatment; CL, control with leptin treatment. Recombinant leptin treatment was for 7 days. The mean SD for eight mice per group are plotted. The bar with letter a is significantly different from the control group at P 0.05. The bars with letter b are significantly different from the obese group at P 0.05. Fig. 2. The effect of leptin on body weight changes in C57BL/6J (control) and ob/ob (leptin-deficient) mice. Mice were weighed at day zero before the start of leptin and saline injection. Body weight measurements were made daily for the next 7 days, and the amount of leptin injected was adjusted accordingly. The mean values were calculated by averaging daily body weight differences for each mouse for all groups. C, control; O, obese; OL, obese with leptin treatment; CL, control with leptin treatment. Leptin treatment was for 7 days. The mean SD for eight mice per group are plotted. The bar with letter a is significantly different from the control group at P 0.05. The bars with letter b are significantly different from the obese group at P 0.05. Fig. 3. The effect of leptin on the major lineages of the marrow in C57BL/6J (control) and ob/ob (leptin-deficient) mice. A composite diagram indicating the proportion of nucleated bone marrow cells of the erythroid, lymphoid, granulocytic, monocytic, and mixed progenitor lineages for each experimental group is presented. Leptin treatment was for 7 days for ob/ob and control mice, whereas analogous groups were given the saline. Values are the mean SD for eight mice per group. *, different from the control or saline-injected group, P 0.05. that represent a substantial reduction in this cell population (Fig. 3). Indeed there was also an overall loss of some 40% of nucleated cells in the marrow of ob/ob mice (Table 1). Taken together, these data showed a 70% decline in the number of cells of the lymphoid lineage in ob/ob compared with control mice (Table 1). This significant decline provides early evidence of the potential importance of leptin to lymphopoiesis. In the case of granulocytic cells, there was no change in their proportion in the marrow of ob/ob mice (Fig. 3). However, there was a 40% decrease in their absolute numbers compared with controls, suggesting a potential role for leptin in their development (Table 1). Conversely, the proportion of cells of the monocytic lineage increased 25% in the ob/ob marrow. Yet they too were affected by the overall decline in numbers of nucleated cells in the marrow of obese mice so that, in actuality, they were 75% the absolute number found in controls (Table 1). Thus, the absolute number of cells of the myeloid lineages was clearly reduced by leptin deficiency. Interestingly, erythroblasts increased in proportion from 18% in control marrow to 30% in ob/ob mice (Fig. 3). However, their absolute numbers remained nearly normal in the absence of leptin (Table 1). The substantial loss in the proportion of lymphoid cells created the apparent increase in the proportion of erythroid cells. This suggests little direct reliance on leptin for the development of red blood cells. Leptin Regulation of Cells of the Lymphoid and Myeloid Lineage in ob/ob Mice. Although 7 days of injection of leptin would seem to constitute a modest degree of intervention, it had dramatic effect on some aspects of hematopoiesis in the ob/ob mice. This was especially true of the lymphoid compartment. The proportion of lymphoid cells in the marrow of leptin-treated ob/ob mice increased 43% (Fig. 3). More importantly, the absolute number of cells increased 80% (Table 1). This represents significant restoration in the numbers of developing lymphocytes in a relatively short period. It adds credence to the idea that leptin plays an important role in promoting or facilitating lymphopoeisis. Leptin supplementation had little impact on the proportion of granulocytes that had remained nearly normal in the untreated obese mice. However, it did result in generation of near-normal numbers of granulocytes in ob/ob mice providing an 80% increase in cells of these lineages (Table 1). In these same mice, addition of leptin also created normal proportions and normal numbers of cells of the monocyte lineage. Thus, a supportive role 2018 www.pnas.org cgi doi 10.1073 pnas.0712053105 Claycombe et al.

Table 1. Cell number in bone marrow Treatment group Nucleated bone marrow ( 10 7 ) Erythroblast B cell number, n Granulocyte Monocyte Progenitor cell, n Control 2.78 0.71 5.20 0.71 7.51 0.52 9.13 0.78 3.26 0.44 2.89 0.15 Obese 1.70 0.46* 4.98 0.56 2.52 0.23* 5.04 0.44* 2.50 0.20* 2.19 0.17* Obese with leptin treatment 2.55 0.52 4.59 0.88 4.64 0.40* 8.97 0.58 3.34 0.29 2.65 0.15* Control with leptin treatment 2.39 0.36 5.39 0.98 5.70 0.37* 6.66 0.97* 2.20 0.21* 1.86 0.09* Nucleated marrow cell numbers are for two femurs. Values are mean SD, n 8. *, different from control, P 0.05. Cell number is determined from mean phenotypic composition and nucleated marrow cell number. for leptin in myelopoietic development also seems probable based on these results. Once again, there appeared to be no real impact of the leptin supplementation on cells of the erythroid lineage (Fig. 3). Whereas the proportion of these cells was normalized in leptin-supplemented obese mice, once again, this was primarily because of the increase in lymphoid and myeloid cells to near-normal levels in the ob/ob. Leptin treatment of the control mice may have constituted an excessive or deleterious amount of this factor because it caused modest shifts in the composition of the marrow of control mice to include a decline in numbers of nucleated cells in the marrow (Fig. 3 and Table 1). Leptin Modulation of Lymphopoiesis in the Marrow. The dramatic reduction in the number and proportion of cells in the lymphoid compartment of the ob/ob mice prompted a more extensive examination of cells of the B lineage that develop primarily in the marrow of adult mammals (23). It is apparent that there was a statistically significant reduction in the proportion of B cells within the marrow of leptin-deficient mice of nearly 50% (Fig. 4A and Table 2). Furthermore the deficiency of leptin also altered the distribution of cells of the B lineage (Fig. 4B and Table 2). Interestingly, there was a 35% reduction in the proportion of pre-b and a 70% reduction in immature B cells in ob/ob mice. Conversely, there was a concomitant increase in pro-b cells, an early B cell lineage, and an increase in the proportion of mature B cells in ob/ob mice. These data suggest that mature lineages may survive the leptin-deficient environment, whereas the suboptimal leptin appeared to limit the ability of pro-b cells to develop into pre-b and immature B cells. The 7-day regimen of leptin injections modestly increased the proportion of pre-b and immature B cells but included a twofold increase in the numbers of pre-b cells and a threefold increase in the number of immature B cells (Table 2). This is indicative of a rapid ramping up and maturation of pro-b cells in the leptin-treated ob/ob mice. As a result, the absolute number of pro-b cells became near normal in numbers (Table 2). It is interesting to note that leptin deficiency had a modest effect on the proportion of so-called mixed progenitor cells (Fig. 3 and Table 1). Perhaps the marked restoration of lymphoid and myeloid cells by the short period of leptin supplementation was also due to the presence of progenitors consisting of a variety of early lineages that rapidly matured on restoration of leptin. Additional Restoration of Lymphopoiesis in ob/ob Mice by 12 Days of Leptin Reconstitution. Although the cellularity of the marrow of the ob/ob mice was nearly normal (91%) after 7 days of addition of leptin, the total B cell population remained at 67%, and the proportion of pre-b cells within the B cell compartment exhibited deficits of 24% (Fig. 4 A and B). We were curious to learn whether further restoration could be accomplished by provision of leptin for 12 days by using methods described for previous studies. However, in this case, controls received vehicle only. Interestingly, by day 12, there were somewhat higher numbers than normal of nucleated cells in the marrow of treated ob/ob mice (3.90 0.6 10 7 nucleated cells) compared with 3.18 0.6 10 7 cells for lean controls (numbers are the average of two femurs per mouse as in Table 1). The 5 days of additional exposure to leptin increased the proportion of CD45R B cells in the marrow to 75% of normal, which fell short of complete restoration [supporting information (SI) Fig. 5]. However, within the B cell compartment there was substantial restoration of the distribution of key cell subsets with pre-b and immature B cells being normal in proportion. The pro-b cells remained somewhat elevated, perhaps an ongoing sign of their ability to survive adverse conditions and serve as an essential precursor population for restoration. Fig. 4. The effect of leptin on the bone marrow B cell populations in C57BL/6J (control) and ob/ob (leptin-deficient) mice. (A) The whole B cell compartment (CD45R ) for each experimental group is shown. (B) The status of B cell subpopulations within each experimental group is shown. C, control; O, obese; OL, obese with leptin treatment; CL, control with leptin treatment. Leptin treatment was for 7 days. The mean SD for eight mice per group are plotted. The bars with letter a are significantly different from the control group at P 0.05. The bars with letter b are significantly different from the obese group at P 0.05. Claycombe et al. PNAS February 12, 2008 vol. 105 no. 6 2019

Table 2. Numbers of B cells by subpopulation Treatment group Pro-B cells ( 10 5 ) Pre-B cells Immature B cells Mature B cells Control 6.16 0.33 4.09 0.25 1.13 0.11 1.13 0.11 Obese 3.87 0.35* 0.88 0.15* 0.14 0.06* 0.95 0.09 Obese with leptin treatment 6.59 0.49 1.89 0.30* 0.45 0.58* 1.37 0.21 Control with leptin treatment 5.88 0.35* 2.97 0.19* 0.78 0.12* 1.11 0.24 *, different from control, P 0.05. Cell number is determined by using the mean phenotypic composition determined for the B cell subpopulation and the calculated number of marrow B cells present for the treatment group. Values are mean SD, n 8. Discussion Finding that leptin bore structural relationship to the IL-6 family was surprising (13). However, it is doubtful that these sequences and structural relationships are by chance. Indeed, many potential immunological roles for leptin are emerging (13, 17). Not unlike the endogenous glucocorticoids that become elevated during stress, which, in turn, alter immune status, especially lymphopoeisis (24), leptin may yet be another molecule that is a molecular connector between the neuroendocrine, metabolic, and immune systems. We will review evidence provided herein that leptin is a critical regulator of food intake and also appears to be critical to lymphopoeisis and myelopoiesis. Remarkably, the leptin-deficient ob/ob mice weighed 2.6 times more than wild-type controls. A mere 7 days of leptin supplementation reduced the diet consumption of these obese mice from6gofdiet per day to just less than the normal amount of 3.2 g of diet per day. Whereas untreated obese mice gained another 3 g, the leptin-treated mice lost 2 g for a weight differential of 5 g in just 7 days. This is a testament to the remarkable ability of leptin to regulate food intake, which was once seen as its primary role. Hematopoiesis proceeds each day providing billions of new leukocytes and RBCs. This obviously requires substantial amounts of nutrients. Indeed, deficits in nutrients, even a single nutrient such as zinc, adversely affects hematopoiesis, especially lymphopoiesis (21, 25). Moreover, suboptimal nutriture has been shown to substantially increase apoptosis among precursor T and B cells (26). Therefore, a link between nutritional status, food intake, and lymphopoiesis has been documented. One might therefore suppose that the consumption of large amounts of nutrients by the obese mouse would provide more than adequate support for hematopoiesis. Yet, in the case of the ob/ob mice, the accompanying deficits in leptin overrode their large intake of diet. For this reason, one of the most striking findings was that these mice had only 60% as many nucleated cells in their marrow as control-type mice (Table 1). Indeed, in the leptin-deficient ob/ob mouse there were losses in cell numbers in all compartments of the marrow except the erythrocyte group. The substantial reduction in the absolute number of cells was most significant in the lymphoid compartment, representing a 70% loss with a sizable or 40% reduction in the granulocytic compartment as well. The 7-day period of injection of leptin increased the number of developing B cells by nearly 80% and generated enough new granulocyte cells so that they were nearly normal in numbers. Thus, even this short period of provision of leptin did considerable restoration, supporting the idea that leptin helps sustain lymphopoiesis and myelopoiesis. By using the markers available to further dissect the events in the B cell compartment of the ob/ob mouse, it was noted that the proportion of pre-b and immature B cells declined very rapidly as the overall numbers of cells in this compartment declined (Table 2 and Fig. 4). Ob/ob mice had 21% the number of pre-b cells and only 12% as many immature B cells as the controls (Table 2). The even greater reduction of immature B cells suggests a sharp decline in the ability to mature in the absence of leptin. However, these two populations of cells also experienced a huge rebound, increasing two- and threefold on leptin supplementation (Fig. 3 and Table 2). This rapid restoration suggests that leptin must be an essential factor in maintaining optimal lymphopoeisis. Finally, it is interesting to note that pro-b cells survived in ob/ob mice reasonably well, which is, no doubt, part of the reason that leptin supplementation of ob/ob mice facilitated a rapid increase in the number of pre-b and immature B cells. Similarly to the early pro-b cells, it is interesting to note that the proportion of mixed progenitors was actually enhanced in ob/ob mice by 30% with only a modest decline in their overall numbers (Fig. 3). Thus, they also seem to be more impervious to the leptin-deficient conditions in the ob/ob mice. The mixed progenitors may have accumulated, in part, due to a reduced ability to move into the myelopoietic and lymphopoietic lines of production. Leptin treatment of the ob/ob mice also normalized the absolute number and proportion of these progenitors. In the dysregulation of hematopoiesis the survival of progenitor cells may allow for rapid regeneration. This may represent a fail-safe mechanism whereby restoration of the immune system can proceed rapidly once conditions improve. Leptin deficiency altered myelopoiesis, especially granulopoiesis. The absolute numbers of cells of the granulocyte series was reduced 40% in the ob/ob mice with a more modest reduction of 25% of the cells of the monocytic lineage (Table 1). However, the proportion of monocytes and granulocytes remained near normal (Fig. 3). As in the case of lymphoid lineage, 7 days of leptin supplementation essentially restored the proportion and absolute number of granulocytes and monocytes to normal (Table 1 and Fig. 3). Thus, leptin also plays a supporting role in myelopoiesis. Substantial additional support for an augmentation role for leptin in thymopoiesis is provided in the literature (19). The ob/ob mice have atrophied thymuses with fewer numbers of thymocytes. Moreover, there was a 10-fold increase in CD4 CD8 pro-t cells in the residual thymus of the leptindeficient mice. This pattern mimics to a degree our findings for pro-b cells in the marrow. After a 12-day period of injection of ob/ob mice with recombinant leptin the numbers of cells in the thymus increased threefold. The number of CD4 CD8 thymocytes increased approximately fourfold to near-normal proportions, whereas the proportion of CD4 CD8 decreased substantially (19). This is also reminiscent of the impact of leptin supplementation on B cell genesis in the marrow of the obese mice found herein. Together the experiments show that leptin promotes or augments lymphopoiesis in the primary immune tissues. It is well known that obesity alters host defense with reduced clearance of pneumonia and high mortality to influenza noted in mouse models (9, 27, 28). Although phagocytosis of Klebsiella pneumoniae was impaired in ob/ob mice, there was no significant decline in numbers of neutrophils or macrophages, which per- 2020 www.pnas.org cgi doi 10.1073 pnas.0712053105 Claycombe et al.

haps is evidence of the greater survival of cells of the myeloid lineages in the marrow (9). How the marrow deficits observed herein affect numbers of circulating B cells and lymphocytes was not determined. Likewise it is not clear whether leptin is directly or indirectly involved in myelopoiesis or lymphopoiesis. Indirectly, leptin could be acting by promoting the production of cytokines or growth-promoting factors by stromal cells. Leptin might also reduce the elevated production of glucocorticoids, cytokines, and inflammatory factors that are prevalent in the ob/ob mouse and other forms of obesity. In the latter case, some of these factors, especially the glucocorticoids, are known to initiate apoptosis among early-lineage cells of the marrow and thymus (24, 26). In vitro experiments that investigate the capacity of leptin to support lymphopoiesis (or myelopoiesis) either alone or in conjunction with cocktails of cytokines might better define the actual role of leptin in lymphopoiesis and myelopoiesis. Materials and Methods Mice. Male C57BL/6J control mice and leptin-deficient obese C57BL/6J-ob/ob (ob/ob) were purchased from The Jackson Laboratory at 2 months of age. Mice were fed Teklad 22/5 rodent diet (Harlan) and provided water ad libitum for 2 weeks before leptin injection. All conditions and experimental protocols were approved by the Michigan State University Committee on Animal Use and Care. Leptin Treatment. Control and ob/ob mice were each divided into two groups of eight mice to be injected with either leptin or vehicle. Leptin-treated mice received daily i.p. injections of 1 g of recombinant leptin protein (Peprotech) per gram of body weight for 7 days. Vehicle-treated mice received a 0.9% saline endotoxin-free solution for 7 days, which was also used for leptin injection. Body weights and food intake were recorded daily with the amount of leptin injected adjusted accordingly. In a smaller experiment, control mice were injected with saline only and ob/ob mice were injected with leptin for a period of 12 days by using the methodology described earlier. Preparation of Bone Marrow Cell Suspensions. Bone marrow was flushed from the femur of each mouse with Harvest buffer that consisted of modified Hank s balanced salt solution, 1 mm Hepes, ph 7.2, and 4% FBS. Red blood cells were removed by lysis and the total number of viable nucleated cells determined by using trypan blue dye exclusion as described in refs. 22 and 24. The cells were suspended in Harvest buffer containing 0.1% sodium azide and placed in ice for phenotypic labeling. Phenotyped samples were fixed in PBS containing 2% formaldehyde, ph7.2, and held on ice until analyzed. Immunophenotypic Labeling of Bone Marrow. For all labeling sets, eight mice were individually analyzed from each of the four treatment groups, e.g., C57BL/J6 controls (saline-injected or leptin-treated), or ob/ob mice (salineinjected or leptin-treated). Employing CD31-AvPECy5 (ERMP12) and Ly6C-FITC (ERMP20 or CD59) were used to divide the marrow into cells of erythroid lineage (CD31 Ly6C ), lymphocytic lineage (CD31 Ly6C ), granulocytes (CD31 Ly6C ), monocytes (CD31 Ly-6C 2 ), and mixed progenitors (CD31 Ly6C ). As per past publications, a third antibody conjugated with phycoerythrin (PE) was used to verify each major lineage group within the marrow, e.g., TER119 for erythrocytes, Ly6G or Gr-1 for granulocytes, and CD45R for lymphoid cells (22, 24). Pro-B cells (CD45 CD43 IgM ), pre-b cells (CD45 CD43 IgM ), and immature mature B cells (CD45 CD43 IgM ) were defined by using anti- CD43 (S7-FITC), anti-igm-pe- and anti-cd45r-percp-cy5.5 (24). Immature/ mature B cells were separately subdivided into immature (IgM IgD ) and mature (IgM IgD ) by using anti-igd-fitc, anti-igm-pe, and anti-cd45r (B220-PerCy5.5). All antibodies were from BD PharMingen (BD Biosciences) except CD31 and Ly6-C that were purchased from Bachem Bioscience. Data Analysis. Statistics-SigmaStat version 3.1 (Systat Software) was used for data analysis. One-way repeated measure ANOVA was used to determine whether a treatment group was significantly different from the control group. If the presence of statistical significance was found, the Holm Sidak method of pairwise multiple comparisons was used to identify treatment groups statistically significantly different from the control or obese group at P 0.05 or greater where n 8 mice per group. All experiments reported herein were performed two or more times, except for reconstitution for 12 days of leptin-deficient ob/ob mice, which was done only once. Standard deviations are reported for all data. 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