A Role for Leptin in Sustaining Lymphopoiesis and Myelopoiesis

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 the addition of G-CSF or GM-CSF did not promote additional many emerging roles in immune function. To better define the role myeloid colony formation. Conversely, when others compared of leptin in hematopoietic processes, a leptin-deficient obese colony formation capacity for wild-type and ob/ob mice, they mouse (ob/ob) and C57BL/6 lean wild-type controls were com- found both produced lymphoid and myeloid colonies, although pared. Despite their large size and consumption of substantial fewer numbers of colonies were produced by marrow from the amounts of nutrients, the ob/ob mice had only 60% as many obese mice (12). Recent evidence that leptin can promote nucleated cells in their marrow as controls. The greatest impact of thymopoiesis has been clearly demonstrated (19). It was found leptin deficiency was on the B cell compartment that had 70% that leptin injection of ob/ob mice that have involuted thymuses fewer cells, reducing the absolute number of pre-B and immature promoted robust thymopoiesis (19). Similarly, the leptin- B cells to 21% and 12% of normal, respectively, and indicating a receptor-defective db/db mice also display atrophied thymuses significant reduction in lymphopoiesis in ob/ob mice. Whereas the and reductions in thymus cell number, indicating a dependency proportion of myeloids remained nearly normal in the obese mice, on leptin (20). they also exhibited a reduction of 40% and 25%, respectively, in We were interested in further exploring the role of leptin in absolute numbers of granulocytes and monocytes. Seven days of lymphopoiesis and myelopoiesis. The experiments described provision of recombinant leptin promoted substantial lymphopoi- herein were performed in vivo comparing and contrasting he- esis, increasing the numbers of B cells in the marrow of the obese matopoietic processes in lean wild-type C57BL/6J mice (control) mice twofold, while doubling and tripling, respectively, the num- versus their mutated ob/ob facsimile that are deficient in func- bers of pre-B and immature B cells. Twelve days of supplementa- tional leptin. In addition, control and ob/ob mice were injected tion brought these subpopulations to near-normal proportions. with recombinant leptin to evaluate its impact on the lympho- Leptin treatment also facilitated myelopoiesis such that the mar- poietic and myelopoietic compartments of the leptin-deficient row of the obese mice contained normal numbers of monocytes mice. The data will show that leptin appears to play an essential and granulocytes after 7 days. Taken together, the data support role sustaining lymphopoiesis and myelopoiesis in the marrow. an important role for leptin in sustaining lymphopoiesis and myelopoiesis. Results Modulation of Body Weight and Food Intake by Leptin. The powerful obesity ͉ ob/ob mouse ͉ pre-B cell ͉ pre-T cell impact of leptin on metabolism, especially food intake, is shown here (Fig. 1). Control mice injected with either leptin or saline eptin is a small 16-KDa protein produced primarily by consumed 3.5 to 3.6 g of diet per day per mouse. The untreated adipocytes (1). It acts on regions of the hypothalamus and ob/ob mice consumed 6.35 g of diet per day per mouse during the L same period and gained an additional4gofbody weight such controls food intake and body temperature (1, 2). The severe Ͼ degree of obesity found in ob/ob mice that are deficient in that their final weight was 2.6 times that of controls (Fig. 2). functional leptin and db/db mice that lack functional leptin Conversely, the 7-day regimen of leptin injections reduced ob/ob intake to near-normal amounts, creating a weight loss of Ϸ2g receptors bears witness to the major role leptin plays in regu- Ͻ lating energy homeostasis and food intake (3, 4). However, these (Fig. 2). Control mice treated with leptin lost 0.5 g of body weight, whereas untreated control mice gained Ͻ1 g of weight two strains of mice also manifest defects in innate and adaptive during the 7-day period. immunity (5, 6). Indeed, the literature demonstrates that leptin can modulate both neuroendocrine (7) and immune functions ϩ Impact of a Deficiency of Leptin on Lymphopoiesis and Myelopoiesis. (6–11). CD34 marrow precursor cells (12), T cells (12), B cells, The next data provide an overview of the impact of obesity and the and phagocytic cells including promyelocytes all bear leptin accompanying deficiencies in leptin on the major lineages devel- receptors, indicating potential modulation of their function or oping in the marrow of the mouse (Fig. 3). By using protocols and metabolism by leptin (7, 13, 14). Interestingly, leptin receptors gating procedures outlined in earlier publications, anti-CD 31 and are members of the class I cytokine receptor family and leptin anti-Ly6C were used to divide the marrow into five major com- itself contains four ␣-helix motifs analogous to IL-6 family partments (21, 22). In addition, anti-Ly6G (Gr-1), anti-CD45R, and members (7, 15). Leptin often is present during acute-phase anti-TER119 were used to further verify the regions containing responses that include C-reactive protein, serum amyloid A, and granulocytic cells, cells of the B lineage, and erythroid lineages, tumor necrosis factor (16, 17). Collectively, this information respectively. Of immediate interest was the 50% decrease in the indicates that leptin modulates a variety of immune functions. proportion of cells in the lymphoid compartment of the ob/ob mice

Evidence that leptin plays some role in maintaining the IMMUNOLOGY integrity of the primary immune tissues has also emerged. However, the literature on the possible roles for leptin in Author contributions: K.J.C., L.E.K., and P.J.F. designed research; K.J.C. and L.E.K. per- hematopoietic functions is limited by being contradictory, as formed research; K.J.C., L.E.K., and P.J.F. analyzed data; and K.J.C. and P.J.F. wrote the pointed out in a past review (13). For example, in one study it paper. was suggested that leptin promotes myelopoiesis to the exclusion The authors declare no conflict of interest. of effects on other lineages in the marrow (18). However, these ‡To whom correspondence should be addressed. E-mail: [email protected]. studies were performed in vitro in serum-free media. The This article contains supporting information online at www.pnas.org/cgi/content/full/ addition of leptin promoted the formation of only a modest 0712053105/DC1. number of granulocyte-macrophage-like colonies. Surprisingly, © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712053105 PNAS ͉ February 12, 2008 ͉ vol. 105 ͉ no. 6 ͉ 2017–2021 Downloaded by guest on September 23, 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 Ϯ SD for eight mice per group are plotted. The bar with letter a is significantly different from the control group at P Ͻ 0.05. The bars with letter b are significantly different from the obese group at P Ͻ 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 impor- tance 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 Fig. 3. The effect of leptin on the major lineages of the marrow in C57BL/6J controls, suggesting a potential role for leptin in their develop- (control) and ob/ob (leptin-deficient) mice. A composite diagram indicating ment (Table 1). Conversely, the proportion of cells of the the proportion of nucleated bone marrow cells of the erythroid, lymphoid, monocytic lineage increased 25% in the ob/ob marrow. Yet they granulocytic, monocytic, and mixed progenitor lineages for each experimen- too were affected by the overall decline in numbers of nucleated tal group is presented. Leptin treatment was for 7 days for ob/ob and control cells in the marrow of obese mice so that, in actuality, they were mice, whereas analogous groups were given the saline. Values are the mean Ϯ Ϸ75% the absolute number found in controls (Table 1). Thus, SD for eight mice per group. *, different from the control or saline-injected the absolute number of cells of the myeloid lineages was clearly group, P Ͻ 0.05. reduced by leptin deficiency. Interestingly, erythroblasts in- creased 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. Fig. 2. The effect of leptin on body weight changes in C57BL/6J (control) and It adds credence to the idea that leptin plays an important role in ob/ob (leptin-deficient) mice. Mice were weighed at day zero before the start promoting or facilitating lymphopoeisis. of leptin and saline injection. Body weight measurements were made daily for Leptin supplementation had little impact on the proportion of the next 7 days, and the amount of leptin injected was adjusted accordingly. granulocytes that had remained nearly normal in the untreated The mean values were calculated by averaging daily body weight differences obese mice. However, it did result in generation of near-normal 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. numbers of granulocytes in ob/ob mice providing an 80% The mean Ϯ SD for eight mice per group are plotted. The bar with letter a is increase in cells of these lineages (Table 1). In these same mice, significantly different from the control group at P Ͻ 0.05. The bars with letter addition of leptin also created normal proportions and normal b are significantly different from the obese group at P Ͻ 0.05. numbers of cells of the monocyte lineage. Thus, a supportive role

2018 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712053105 Claycombe et al. Downloaded by guest on September 23, 2021 Table 1. Cell number in bone marrow Nucleated bone Erythroblast B cell Granulocyte Monocyte Progenitor marrow cells, n cells, n number, n cells, n cells, n cell, n Treatment group (ϫ 107)† (ϫ 106)‡ (ϫ 106)‡ (ϫ 106)‡ (ϫ 106)‡ (ϫ 106)‡

Control 2.78 Ϯ 0.71 5.20 Ϯ 0.71 7.51 Ϯ 0.52 9.13 Ϯ 0.78 3.26 Ϯ 0.44 2.89 Ϯ 0.15 Obese 1.70 Ϯ 0.46* 4.98 Ϯ 0.56 2.52 Ϯ 0.23* 5.04 Ϯ 0.44* 2.50 Ϯ 0.20* 2.19 Ϯ 0.17* Obese with leptin treatment 2.55 Ϯ 0.52 4.59 Ϯ 0.88 4.64 Ϯ 0.40* 8.97 Ϯ 0.58 3.34 Ϯ 0.29 2.65 Ϯ 0.15* Control with leptin treatment 2.39 Ϯ 0.36 5.39 Ϯ 0.98 5.70 Ϯ 0.37* 6.66 Ϯ 0.97* 2.20 Ϯ 0.21* 1.86 Ϯ 0.09*

†Nucleated marrow cell numbers are for two femurs. Values are mean Ϯ SD, n ϭ 8. *, different from control, P Յ 0.05. ‡Cell number is determined from mean phenotypic composition and nucleated marrow cell number.

for leptin in myelopoietic development also seems probable than normal of nucleated cells in the marrow of treated ob/ob based on these results. Once again, there appeared to be no real mice (3.90 Ϯ 0.6 ϫ 107 nucleated cells) compared with 3.18 Ϯ impact of the leptin supplementation on cells of the erythroid 0.6 ϫ 107 cells for lean controls (numbers are the average of two lineage (Fig. 3). Whereas the proportion of these cells was femurs per mouse as in Table 1). The 5 days of additional normalized in leptin-supplemented obese mice, once again, this exposure to leptin increased the proportion of CD45Rϩ B cells was primarily because of the increase in lymphoid and myeloid in the marrow to 75% of normal, which fell short of complete cells to near-normal levels in the ob/ob. restoration [supporting information (SI) Fig. 5]. However, within Leptin treatment of the control mice may have constituted an the B cell compartment there was substantial restoration of the excessive or deleterious amount of this factor because it caused distribution of key cell subsets with pre-B and immature B cells modest shifts in the composition of the marrow of control mice being normal in proportion. The pro-B cells remained somewhat to include a decline in numbers of nucleated cells in the marrow elevated, perhaps an ongoing sign of their ability to survive (Fig. 3 and Table 1). adverse conditions and serve as an essential precursor popula- tion for restoration. 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 environ- ment, 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 pro- portion 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 IMMUNOLOGY Leptin Reconstitution. Although the cellularity of the marrow of the ob/ob mice was nearly normal (91%) after 7 days of addition Fig. 4. The effect of leptin on the bone marrow B cell populations in C57BL/6J of leptin, the total B cell population remained at 67%, and the (control) and ob/ob (leptin-deficient) mice. (A) The whole B cell compartment ϩ proportion of pre-B cells within the B cell compartment exhib- (CD45R ) for each experimental group is shown. (B) The status of B cell ited deficits of 24% (Fig. 4 A and B). We were curious to learn subpopulations within each experimental group is shown. C, control; O, obese; OL, obese with leptin treatment; CL, control with leptin treatment. whether further restoration could be accomplished by provision Leptin treatment was for 7 days. The mean Ϯ SD for eight mice per group are of leptin for 12 days by using methods described for previous plotted. The bars with letter a are significantly different from the control studies. However, in this case, controls received vehicle only. group at P Ͻ 0.05. The bars with letter b are significantly different from the Interestingly, by day 12, there were somewhat higher numbers obese group at P Ͻ 0.05.

Claycombe et al. PNAS ͉ February 12, 2008 ͉ vol. 105 ͉ no. 6 ͉ 2019 Downloaded by guest on September 23, 2021 Table 2. Numbers of B cells by subpopulation Pro-B cells Pre-B cells Immature B cells Mature B cells Treatment group (ϫ 105)† (ϫ 106)† (ϫ 106)† (ϫ 106)†

Control 6.16 Ϯ 0.33 4.09 Ϯ 0.25 1.13 Ϯ 0.11 1.13 Ϯ 0.11 Obese 3.87 Ϯ 0.35* 0.88 Ϯ 0.15* 0.14 Ϯ 0.06* 0.95 Ϯ 0.09 Obese with leptin treatment 6.59 Ϯ 0.49 1.89 Ϯ 0.30* 0.45 Ϯ 0.58* 1.37 Ϯ 0.21 Control with leptin treatment 5.88 Ϯ 0.35* 2.97 Ϯ 0.19* 0.78 Ϯ 0.12* 1.11 Ϯ 0.24

*, different from control, P Յ 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 Ϯ SD, n ϭ 8.

Discussion suggests a sharp decline in the ability to mature in the absence Finding that leptin bore structural relationship to the IL-6 family of leptin. However, these two populations of cells also experi- was surprising (13). However, it is doubtful that these sequences enced a huge rebound, increasing two- and threefold on leptin and structural relationships are by chance. Indeed, many poten- supplementation (Fig. 3 and Table 2). This rapid restoration tial immunological roles for leptin are emerging (13, 17). Not suggests that leptin must be an essential factor in maintaining unlike the endogenous glucocorticoids that become elevated optimal lymphopoeisis. Finally, it is interesting to note that pro-B during stress, which, in turn, alter immune status, especially cells survived in ob/ob mice reasonably well, which is, no doubt, lymphopoeisis (24), leptin may yet be another molecule that is part of the reason that leptin supplementation of ob/ob mice a molecular connector between the neuroendocrine, metabolic, facilitated a rapid increase in the number of pre-B and immature and immune systems. We will review evidence provided herein B cells. that leptin is a critical regulator of food intake and also appears Similarly to the early pro-B cells, it is interesting to note that to be critical to lymphopoeisis and myelopoiesis. the proportion of mixed progenitors was actually enhanced in Remarkably, the leptin-deficient ob/ob mice weighed 2.6 times ob/ob mice by 30% with only a modest decline in their overall more than wild-type controls. A mere 7 days of leptin supple- numbers (Fig. 3). Thus, they also seem to be more impervious mentation reduced the diet consumption of these obese mice to the leptin-deficient conditions in the ob/ob mice. The mixed from6gofdiet per day to just less than the normal amount of progenitors may have accumulated, in part, due to a reduced 3.2 g of diet per day. Whereas untreated obese mice gained ability to move into the myelopoietic and lymphopoietic lines of another 3 g, the leptin-treated mice lost 2 g for a weight production. Leptin treatment of the ob/ob mice also normalized differential of Ϸ5 g in just 7 days. This is a testament to the the absolute number and proportion of these progenitors. In the remarkable ability of leptin to regulate food intake, which was dysregulation of hematopoiesis the survival of progenitor cells once seen as its primary role. may allow for rapid regeneration. This may represent a fail-safe Hematopoiesis proceeds each day providing billions of new mechanism whereby restoration of the immune system can leukocytes and RBCs. This obviously requires substantial proceed rapidly once conditions improve. amounts of nutrients. Indeed, deficits in nutrients, even a single Leptin deficiency altered myelopoiesis, especially granulopoi- nutrient such as zinc, adversely affects hematopoiesis, especially esis. The absolute numbers of cells of the granulocyte series was lymphopoiesis (21, 25). Moreover, suboptimal nutriture has been reduced Ϸ40% in the ob/ob mice with a more modest reduction shown to substantially increase apoptosis among precursor T and of 25% of the cells of the monocytic lineage (Table 1). However, B cells (26). Therefore, a link between nutritional status, food the proportion of monocytes and granulocytes remained near intake, and lymphopoiesis has been documented. One might normal (Fig. 3). As in the case of lymphoid lineage, 7 days of therefore suppose that the consumption of large amounts of leptin supplementation essentially restored the proportion and nutrients by the obese mouse would provide more than adequate absolute number of granulocytes and monocytes to normal support for hematopoiesis. Yet, in the case of the ob/ob mice, the (Table 1 and Fig. 3). Thus, leptin also plays a supporting role in accompanying deficits in leptin overrode their large intake of myelopoiesis. diet. For this reason, one of the most striking findings was that Substantial additional support for an augmentation role for these mice had only 60% as many nucleated cells in their marrow leptin in thymopoiesis is provided in the literature (19). The as control-type mice (Table 1). Indeed, in the leptin-deficient ob/ob mice have atrophied thymuses with fewer numbers of ob/ob mouse there were losses in cell numbers in all compart- thymocytes. Moreover, there was a 10-fold increase in ments of the marrow except the erythrocyte group. The sub- CD4ϪCD8Ϫ pro-T cells in the residual thymus of the leptin- stantial reduction in the absolute number of cells was most deficient mice. This pattern mimics to a degree our findings for significant in the lymphoid compartment, representing a 70% pro-B cells in the marrow. After a 12-day period of injection of loss with a sizable or 40% reduction in the granulocytic com- ob/ob mice with recombinant leptin the numbers of cells in the partment as well. The 7-day period of injection of leptin in- thymus increased threefold. The number of CD4ϩCD8ϩ thymo- creased the number of developing B cells by nearly 80% and cytes increased approximately fourfold to near-normal propor- generated enough new granulocyte cells so that they were nearly tions, whereas the proportion of CD4ϪCD8Ϫ decreased substan- normal in numbers. Thus, even this short period of provision of tially (19). This is also reminiscent of the impact of leptin leptin did considerable restoration, supporting the idea that supplementation on B cell genesis in the marrow of the obese leptin helps sustain lymphopoiesis and myelopoiesis. mice found herein. Together the experiments show that leptin By using the markers available to further dissect the events in promotes or augments lymphopoiesis in the primary immune the B cell compartment of the ob/ob mouse, it was noted that the tissues. proportion of pre-B and immature B cells declined very rapidly It is well known that obesity alters host defense with reduced as the overall numbers of cells in this compartment declined clearance of pneumonia and high mortality to influenza noted in (Table 2 and Fig. 4). Ob/ob mice had Ϸ21% the number of pre-B mouse models (9, 27, 28). Although phagocytosis of Klebsiella cells and only 12% as many immature B cells as the controls pneumoniae was impaired in ob/ob mice, there was no significant (Table 2). The even greater reduction of immature B cells decline in numbers of neutrophils or macrophages, which per-

2020 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712053105 Claycombe et al. Downloaded by guest on September 23, 2021 haps is evidence of the greater survival of cells of the myeloid Hank’s balanced salt solution, 1 mM Hepes, pH 7.2, and 4% FBS. Red blood cells lineages in the marrow (9). How the marrow deficits observed were removed by lysis and the total number of viable nucleated cells deter- herein affect numbers of circulating B cells and lymphocytes was mined 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 not determined. Likewise it is not clear whether leptin is directly placed in ice for phenotypic labeling. Phenotyped samples were fixed in PBS or indirectly involved in myelopoiesis or lymphopoiesis. Indi- containing 2% formaldehyde, pH7.2, and held on ice until analyzed. rectly, leptin could be acting by promoting the production of cytokines or growth-promoting factors by stromal cells. Leptin Immunophenotypic Labeling of Bone Marrow. For all labeling sets, eight mice might also reduce the elevated production of glucocorticoids, were individually analyzed from each of the four treatment groups, e.g., cytokines, and inflammatory factors that are prevalent in the C57BL/J6 controls (saline-injected or leptin-treated), or ob/ob mice (saline- ob/ob mouse and other forms of obesity. In the latter case, some injected or leptin-treated). Employing CD31-AvPECy5 (ERMP12) and Ly6C-FITC (ERMP20 or CD59) were used to divide the marrow into cells of erythroid of these factors, especially the glucocorticoids, are known to Ϫ Ϫ ϩ Ϫ initiate apoptosis among early-lineage cells of the marrow and lineage (CD31 Ly6C ), lymphocytic lineage (CD31 Ly6C ), granulocytes (CD31Ϫ Ly6Cϩ), monocytes (CD31Ϫ Ly-6C2ϩ), and mixed progenitors (CD31ϩ thymus (24, 26). In vitro experiments that investigate the capacity Ly6Cϩ). As per past publications, a third antibody conjugated with phyco- of leptin to support lymphopoiesis (or myelopoiesis) either alone erythrin (PE) was used to verify each major lineage group within the marrow, or in conjunction with cocktails of cytokines might better define e.g., TER119 for erythrocytes, Ly6G or Gr-1 for granulocytes, and CD45R for the actual role of leptin in lymphopoiesis and myelopoiesis. lymphoid cells (22, 24). Pro-B cells (CD45ϩ CD43ϩ IgMϪ), pre-B cells (CD45ϩ CD43Ϫ IgMϪ), and Materials and Methods immature–mature B cells (CD45ϩ CD43Ϫ IgMϩ) were defined by using anti- CD43 (S7-FITC), anti-IgM-PE- and anti-CD45R-PerCP-Cy5.5 (24). Immature/ Mice. Male C57BL/6J control mice and leptin-deficient obese C57BL/6J-ob/ob ϩ Ϫ (ob/ob) were purchased from The Jackson Laboratory at 2 months of age. Mice mature B cells were separately subdivided into immature (IgM IgD ) and mature (IgMϩ IgDϩ) by using anti-IgD-FITC, anti-IgM-PE, and anti-CD45R were fed Teklad 22/5 rodent diet (Harlan) and provided water ad libitum for (B220-PerCy5.5). All antibodies were from BD PharMingen (BD Biosciences) 2 weeks before leptin injection. All conditions and experimental protocols except CD31 and Ly6-C that were purchased from Bachem Bioscience. were approved by the Michigan State University Committee on Animal Use and Care. Data Analysis. Statistics-SigmaStat version 3.1 (Systat Software) was used for data analysis. One-way repeated measure ANOVA was used to determine Leptin Treatment. Control and ob/ob mice were each divided into two groups whether a treatment group was significantly different from the control of eight mice to be injected with either leptin or vehicle. Leptin-treated mice group. If the presence of statistical significance was found, the Holm–Sidak received daily i.p. injections of 1 ␮g of recombinant leptin protein (Peprotech) method of pairwise multiple comparisons was used to identify treatment per gram of body weight for 7 days. Vehicle-treated mice received a 0.9% groups statistically significantly different from the control or obese group at saline endotoxin-free solution for 7 days, which was also used for leptin P Յ 0.05 or greater where n ϭ 8 mice per group. All experiments reported injection. Body weights and food intake were recorded daily with the amount herein were performed two or more times, except for reconstitution for 12 of leptin injected adjusted accordingly. In a smaller experiment, control mice days of leptin-deficient ob/ob mice, which was done only once. Standard were injected with saline only and ob/ob mice were injected with leptin for a deviations are reported for all data. period of 12 days by using the methodology described earlier. ACKNOWLEDGMENTS. We thank Joe Frentzel and Dr. Mark Trottier for their Preparation of Bone Marrow Cell Suspensions. Bone marrow was flushed from technical assistance. This work was supported by National Institutes of Health the femur of each mouse with Harvest buffer that consisted of modified Grant DK 52289.

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