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Clinical Endocrinology Branch (E.A.O., E.D.J., E.K.C., J.R.Y., P.G.), National Institute of Diabetes, Digestive, and Kidney Diseases, and Laboratory of Clinical Infectious Diseases (L.D., G.U., S.M.H.), National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892; and Amgen, Inc. (A.M.D.), Thousand Oaks, California 91320
Address all correspondence and requests for reprints to: Phillip Gorden, M.D., 10 Center Drive, MSC 1612, Room CRC 65940, Bethesda, Maryland 20892-1612. E-mail: PhillipG{at}intra.niddk.nih.gov.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Objective: Our objective was to study lymphocyte subpopulations and in vitro peripheral blood mononuclear cell (PBMC) activation during a study evaluating the effects of leptin on metabolic functions in severe lipodystrophy (serum leptin levels < 4 ng/ml).
Design and Setting: We conducted an open-label study with patients serving as their own control at the Clinical Research Center of the National Institutes of Health.
Patients: Ten patients (age range, 15–63 yr; one male and nine females) with generalized forms of lipodystrophy were studied.
Intervention: Patients were treated with recombinant human leptin to achieve high normal concentrations for 4 to 8 months.
Results: Leptin levels increased from 1.8 ± 0.4 to 16.5 ± 3.9 ng/dl (P < 0.001), whereas metabolic control improved [glycosylated hemoglobin (HbA1c) fell from 9.3 ± 0.4 to 7.1 ± 1.4%, P < 0.001, and triglycerides decreased by 45 ± 11% from a mean of 1490 ± 710 mg/dl, P = 0.001]. Lymphocyte subsets were studied by flow cytometry at baseline and at 4 and 8 months of therapy. PBMC responsiveness was evaluated by cytokine release and proliferation after stimulation with phytohemagglutinin, phytohemagglutinin plus IL-12, lipopolysaccharide, and lipopolysaccharide plus interferon-
at baseline and 4 months. Various T lymphocyte subsets were significantly lower than age- and sex-matched controls at baseline; however, the CD4/CD8 ratio was normal. The relative percentages of B lymphocytes and monocytes were elevated, although the absolute levels were normal. Leptin therapy induced significant changes in T lymphocyte subsets, which normalized both the absolute number of T lymphocyte subsets and relative percentages of all lineages. Additionally, in vitro TNF-
secreted from PBMC of patients was significantly increased to normal after 4 months of leptin therapy compared with baseline.
Conclusion: These data support existing evidence that leptin has a modest immunomodulatory effect in hypoleptinemic humans.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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and increased production of IL-4 (2). Exogenous restoration of leptin to normal levels by injection reverses these abnormalities (2, 3). Data regarding the immune modulatory actions of leptin in humans are extremely limited. Although malnourished humans are noted to have hypoleptinemia along with immune abnormalities similar to those found in starved animals, there has been no direct evidence that leptin mediates the reversal of these immunological abnormalities.
Leptin replacement in leptin deficiency states has facilitated understanding the physiological importance of this hormone in humans. Congenital absence of leptin as a result of leptin mutations and severe lipodystrophy, characterized by a paucity of fat cells making leptin, are conditions in which restorative leptin therapy has been shown to be effective in correcting multiple metabolic and hormonal abnormalities (4, 5, 6, 7). Farooqi et al. (8) reported one patient with a leptin mutation and low T lymphocyte subsets. They also found decreased peripheral blood mononuclear cell (PBMC) cytokine secretion in two patients. Recombinant methionyl human leptin (r-metHuLeptin) therapy normalized these abnormalities. Furthermore, Chan et al. (9) pointed out that a more chronic leptin deficiency and leptin exposure is required to demonstrate an effect on lymphocyte and cytokine levels. Additionally, Ozata et al. (10) noted excess early mortality in a consanguineous Turkish pedigree carrying a leptin mutation and hypothesized that impaired T cell immunity might contribute to infection susceptibility.
In this study, we have examined immunological features of patients with severe lipodystrophy and low leptin levels. We studied patients at their preleptin therapy at baseline and after their circulating leptin levels had been normalized for 4 and 8 months.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We studied lymphocyte subpopulations and in vitro PBMC cytokine production and response during r-metHuLeptin therapy of severe lipodystrophy. The study was an open-label, prospective study in which each subject’s data were compared with his/her baseline. The institutional review board of the National Institute of Diabetes, Digestive, and Kidney Diseases approved the study. Patients or their legal guardians signed informed consent.
Patients
Ten patients (one male and nine females; age range, 15–63 yr; six with congenital generalized, two with acquired generalized, and two with Dunnigan’s familial partial lipodystrophy) were enrolled. The gender, ages, and types of lipodystrophy are shown in Table 1
. All patients with congenital generalized lipodystrophy had mutations in AGPAT-2. The two patients with Dunnigan’s familial partial lipodystrophy had mutations in Lamin A/C. All patients had low circulating leptin levels and metabolic abnormalities such as diabetes mellitus and hypertriglyceridemia (Table 1).
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The r-metHuLeptin therapy was given as a self-administered, twice-daily sc injection as previously described (4). The dose was escalated to the full dose over the first 2 months of treatment. Thereafter, the usual replacement dose was 0.06–0.08 mg/kg·d for females and 0.04 mg/kg·d for males in an attempt to simulate the normal to high physiological range. Patients were evaluated at the Clinical Research Center of the National Institutes of Health at baseline, 4 months, and 8 months. Inpatient data were collected on a metabolic unit during each visit. At baseline, patients were on aggressive conventional treatments for diabetes and dyslipidemia. These medications were subsequently lowered or discontinued if indicated.
Lymphocyte subset analysis
Lymphocyte subsets were analyzed at baseline and at 4 months for all patients as well as at 8 months for a subset of these. Flow cytometry was performed using a FACS scan flow cytometer and appropriate directly conjugated monoclonal antibodies (Becton Dickinson-PharMingen, San Diego, CA). An institutional normal control range was predetermined using data from 30 normal volunteers ranging from 15–75 yr old; these data were consistent with observations published previously. Analyses were performed on fresh lymphocytes at the time of sampling.
Proliferation response and cytokine release from PBMC
In vitro PBMC responsiveness was evaluated by cytokine release and proliferation after stimulation with lipopolysaccharide (LPS), LPS plus IFN-, phytohemagglutinin (PHA), and PHA plus IL-12 at baseline and 4 months. PBMC were prepared from heparinized whole blood within 24 h of phlebotomy by density gradient separation, and 106 cells/ml were plated in 1 ml complete RPMI (11). Selected wells were stimulated with PHA 1:100 (Life Technologies, Rockville, MD); Escherichia coli-derived LPS, 200 ng/ml (Sigma Chemical Co., St. Louis, MO); LPS plus IFN-, 1000 U/ml (R&D Systems, Minneapolis, MN); and PHA plus IL-12 p70 heterodimer, 1 ng/ml (R&D Systems). PBMC were stimulated for 48 h at 37 C in 5% CO2; culture supernatants were aliquoted and frozen at –20 C for later cytokine determinations. Samples were thawed once and examined for IFN- and TNF- secretion. Cytokine concentrations were determined in duplicate by ELISA (R&D Systems) as specified by the manufacturer. All cytokine determinations were done with the same lots of reagents. For proliferation studies, unstimulated and PHA-stimulated wells (2 x 105 PBMC per well) were labeled in triplicate in the presence of [3H]thymidine for the last 8 h of a 48-h incubation. Tritiated cells were harvested onto fiberglass filters and quantitated on a Hewlett-Packard Top Count, as described (12).
Controls for the PBMC studies
On the days that samples were obtained from study patients, blood was drawn from one or two age- and sex-matched controls who had no known medical conditions and who were on no medications or supplements. PBMC were isolated and subjected to the same procedure as described above. Control experiments were carried out simultaneously to the patient studies, and cytokine determinations for each paired set of patient and control samples were done with the same lots of reagents. Control samples were randomly placed on the same plates with patient samples.
In vitro leptin exposure
To determine whether there were any in vitro effects of r-metHuLeptin on PBMC cytokine response or production, we prepared patient or normal donor PBMC as above and incubated them with varying concentrations of the same r-metHuLeptin preparation as was used for therapy, up to 100 ng/ml. The r-metHuLeptin was added to PBMC preparations at the same time as the other stimuli and incubated as above. After 48 h, supernatants were harvested, aliquoted, and frozen at –20 C. For analysis, samples were thawed and assayed for IL-1b, IL-6, IL-10, IL-12, TNF-, and IFN- using the Bio-Rad Bioplex system on a Bio-Rad Bioplex reader according to the manufacturer’s directions (Bio-Rad Laboratories, Hercules, CA).
Biochemical analyses
Serum leptin levels were determined by immunoassays with the use of a commercial kit (Linco Research, St. Charles, MO). Glycosylated hemoglobin (HbA1c) values were measured by ion-exchange HPLC (Bio-Rad). Serum glucose and lipid values were determined according to standard methods with the use of automated equipment (Beckman, Fullerton, CA). Samples were drawn after an overnight fast at least 8 h after the previously administered dose of r-metHuLeptin.
Statistical analyses
Measurements are presented as mean ± SE To compare study variables during various study periods, ANOVA with repeated measures was used. Paired t test was employed to compare baseline data with various time points wherever applicable. Nonparametric Mann-Whitney rank sum test was used to compare the percentages of lymphocytes where the variables did not follow a normal distribution. A P value < 0.05 was accepted as statistically significant.
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Results |
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All patients were hypoleptinemic and had metabolic abnormalities such as diabetes mellitus and hypertriglyceridemia (Table 1
). None of the patients reported in this paper were known to have or to have had a history of recurrent or severe skin, respiratory, or other systemic infections or opportunistic infections.
Metabolic and hormonal changes during therapy
Throughout the study, circulating leptin levels increased from 1.8 ± 0.4 ng/ml to 16.5 ± 3.9 ng/ml (P < 0.001), whereas metabolic control dramatically improved as evidenced by HbA1c falling from 9.3 ± 0.4 to 7.1 ± 1.4% (P < 0.001) and triglycerides decreasing by 45 ± 11% from a mean of 1490 ± 710 mg/dl (P = 0.001). Additional hormonal, metabolic, and body composition changes have been reported previously (4, 5, 6, 7, 13).
Lymphocyte subsets at baseline
The leptin-deficient lipodystrophic patients had relatively higher percentage of B lymphocytes (patients, 19.0 ± 3.0%; control, 4.8–15.9%) and absolute numbers of B lymphocytes than controls (patients, 434 ± 119; control, 88–330). The absolute number and percentage of T cells were in the normal range (Table 2), although the percentage of T cells was close to the lower limit of normal (data not shown). As shown in Table 2, the CD4/CD8 ratio was 1.74 ± 0.09. The number and relative percentages of both of these subsets were in the normal range.
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. The relative percentages of these were within the range seen in healthy controls (data not shown).
Changes in lymphocyte subsets with therapy (Table 2)
The r-metHuLeptin therapy caused a significant increase in the absolute number of T cells. This occurred in essentially all lineages. The number of both CD4 and CD8 cells increased, with no significant change in the CD4/CD8 ratio.
Lymphocytes coexpressing CD3 along with either HLA-DR or CD25 are considered to be activated T lymphocytes. Although the number and percentage of these cells were in the normal range at baseline and stayed inside this range with therapy, there was a modest increase in the number of CD3+/CD25+ cells (Table 2). The number of cells carrying the natural killer (NK) markers CD16 or CD56 remained the same from the baseline to 4 months.
Although the number of T cells increased significantly, there were no changes in the absolute number of B cells (433 ± 117). This led to the near normalization of the high B cell percentage observed at baseline (4 months, 16.6 ± 2.4%; control, 4.8–15.9%). The changes noted at 4 months were sustained at 8 months (data not shown). Likewise, leptin replacement led to a fall in the percentage of CD40+ monocytes (baseline, 41.0 ± 8.2%; 4 months, 24.7 ± 5.3%; 8 months, 22.6 ± 5.9%).
In vitro PBMC functional studies
Having noted changes in the number and surface markers of both circulating lymphocytes and monocytes, we examined further the effects of in vivo r-metHuLeptin therapy on in vitro cell function using a standard set of stimulation conditions that are sensitive to lymphocyte and monocyte function as well as their interaction. Spontaneous cell proliferation was higher than controls at baseline (741 ± 209 cpm; control, 427 ± 38 cpm; P = 0.048) and normalized after 4 months of r-metHuLeptin therapy (401 ± 56 cpm). PHA-stimulated proliferation, however, was normal at baseline (34,085 ± 4,411 cpm) and at 4 months of therapy (44,073 ± 7,716 cpm) (control, 40,427 ± 5,017 cpm).
Both unstimulated and stimulated TNF- production was low in patients with untreated hypoleptinemia (Fig. 1). In contrast, r-metHuLeptin therapy normalized TNF- production after stimulation with predominantly T cell (PHA) or monocyte (LPS and LPS plus IFN-) agonists. The most robust increase in TNF- production came in response to LPS and LPS plus IFN-, suggesting that r-metHuLeptin therapy may be affecting responses mediated through the Toll-like receptor pathway (Fig. 1).
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production and IFN- production and response, we examined the production of IFN- in response to PHA and PHA plus IL-12 (Fig. 2). Neither resting nor PHA-stimulated levels of IFN- production were significantly altered by r-metHuLeptin, nor were they different from controls. However, the costimulatory effect of IL-12 on the release of IFN- was attenuated after r-metHuLeptin therapy (Fig. 2C).
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production in response to LPS and IFN- could be indirect, as a result of improved metabolic regulation, or a direct effect of leptin on cellular cytokine production and response. In an attempt to distinguish these possibilities, we incubated normal PBMC with varying doses of r-metHuLeptin in vitro to determine whether the addition of r-metHuLeptin similar to levels that we could measure in vivo had a direct measurable effect. These were normal cells obtained from individuals who did not have metabolic abnormalities, had normal fat distribution, and were presumed to have normal leptin levels. In our 48-h assay using the same stimuli as for patient cells, we could not detect a significant effect of short-term r-metHuLeptin incubation on cytokine production or response. In particular, TNF- production in response to LPS was not augmented. There were also no significant effects seen with IL-1, IL-6, IL-10, IL-12, or IFN- (data not shown). Potential differences from in vitro studies (14) could be a result of technical differences and/or differences in leptin concentrations. |
Discussion |
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secretion in vitro. C-reactive protein levels have been previously shown to be unchanged by r-metHuLeptin therapy in these patients (7). There are limitations to our study. The primary end-point of the study was an effect of r-metHuLeptin on glucose and lipid control; the immune parameters were measured as ancillary end-points. Second, although the reported immunological parameters were being collected, there were simultaneous changes occurring in metabolic and other hormonal factors; glucose and lipid control improved dramatically, insulin sensitivity significantly increased, tissue deposition of triglycerides was reduced, and impaired menstrual function was restored. All these simultaneous changes may have direct or indirect effects on immune function. However, whatever these critical underlying changes are, they were easily demonstrated in cells ex vivo that were not incubated in autologous plasma. Thus, our data provide the largest collection available from patients with an acquired leptin deficiency.
Leptin and lymphocyte function: current evidence and our observations
Consistently, ob/ob and db/db mice have a marked reduction in the size and cellularity of the thymus and exhibit defective T-cell-mediated immunity (2, 3). Furthermore, starvation and malnutrition, two conditions characterized by low leptin levels, are also associated with alterations of the immune response and thymic atrophy, which can be reversed by leptin administration (15, 16). The connection between leptin deficiency and in vivo immune defects in rodent models, however, is likely to be more complex. The ob/ob and db/db mice display multiple endocrine and metabolic modifications, including hypercorticosteronemia and diabetes, which may indirectly affect the immune system. Similarly, leptin deficiency after starvation in rodents is linked to increased glucocorticoid levels and decreased levels of thyroid hormone and GH, each of which may mediate immune suppression (17). Both the direct and indirect effects of leptin are thus likely to account for the immune defects observed in leptin-deficient animals. In human congenital leptin deficiency, one patient was reported with decreased numbers of circulating CD4+ T cells and impaired T cell proliferation and cytokine release, all of which were reversed by the administration of recombinant leptin (8).
In contrast to the single patient reported with congenital leptin deficiency, our patients with lipodystrophy and low leptin levels did not have markedly low CD4+ T lymphocyte counts. However, after r-metHuLeptin therapy, we observed a significant increase in both CD4+ and CD8+ T cells, whereas the CD4/CD8 ratio was preserved. Although NK T cells (CD3+/CD16/56+) also increased in number during r-metHuLeptin therapy, classical NK cells (CD3–/CD16/56+) did not. All CD3+ T cell numbers increased during r-metHuLeptin therapy, whereas B cell numbers remained unchanged, thereby correcting the abnormally high percentage of B cells at baseline into the normal range. These data suggest that leptin has a trophic effect on CD3+ T lymphocytes but not B cells. It is important to note that the long isoform of the leptin receptor (OB-Rb) is expressed on both T and B cells, suggesting that leptin may exert its effect directly (15, 16, 18). Leptin has been reported to stimulate the proliferation of T cells in vitro, to promote T helper-1 responses, and to protect T cells from corticosteroid-induced apoptosis in rodents (15, 16).
Leptin and autoimmune inflammatory conditions: current evidence and our observations
The above-mentioned immunomodulatory and T-helper-1-promoting effects suggest that leptin may play a part in the regulation of inflammatory conditions. An important question remains as to whether leptin may induce or exacerbate autoimmune conditions. Consistently, leptin-deficient mice are protected from inflammation mediated by T and B cells in different disease models, including experimental autoimmune encephalomyelitis, type 1 diabetes, experimental colitis, and antigen-induced arthritis (18, 19, 20, 21). Administration of exogenous leptin restores the responsiveness of ob/ob mice to T-cell-activating stimuli (19). For example, in experimental autoimmune encephalomyelitis in animals with leptin deficiency, replacement of leptin after disease onset enables the expected onset of symptoms (22). Similarly, in nonobese diabetic mice, leptin administration enables the expected autoimmune destruction of pancreatic ß-cells and increases IFN- production by peripheral T cells.
To dissect the role of leptin in autoimmune disease in humans is complex because of the variability of this group of diseases and the contributions of environment. The conditions associated with physiological leptin deficiency are so rare that it is difficult to assess whether these patients are actually protected from autoimmune diseases. From our observations, it does not appear that leptin deficiency offers any protection to these patients. We have observed a high background of autoimmune disease in patients with both congenital and acquired forms of lipodystrophy including thyroid disease, nephropathies, autoimmune hepatitis, and type 1 diabetes.
Our data suggest that markers of PBMC activation can be induced after leptin replacement therapy. Within the time frame of observation covered in this paper, we did not note significant changes in the prevalence of clinical markers of autoimmunity, such as the presence of new autoantibodies. Furthermore, administration of pharmacological doses of leptin to non-leptin-deficient subjects has shown essentially no elevation of cytokines or inflammatory markers (23, 24). However, we recently reported two cases of acquired generalized lipodystrophy who developed significant worsening of proteinuria while receiving r-metHuLeptin therapy (25). Their kidney biopsies showed membranoproliferative glomerulonephritis. We cannot exclude the possibility that leptin may have contributed to the worsening kidney disease in these patients.
Leptin and inflammation: current evidence and our observations
The innate immune system has a major role in the regulation of leptin production. In experimental animal models, leptin levels are acutely increased by inflammatory and infectious stimuli, such as LPS, turpentine, and proinflammatory cytokines (26, 27). The increase in leptin production during infection and inflammation strongly suggests that leptin is part of a cytokine cascade that orchestrates the innate immune response and host defense mechanisms. However, both pro- and antiinflammatory effects have been described for leptin depending on the experimental model investigated.
In vitro, leptin stimulates both pro- and antiinflammatory cytokine production in monocytes and macrophages (28, 29, 30, 31). Macrophages isolated from ob/ob mice show increased basal expression of IL-6 and seem to be constitutively activated, implying that leptin may inhibit macrophage activation in vivo (32). However, ob/ob mice also display impaired innate host response to bacterial pneumonia, suggesting that leptin plays an important part in host defense against infection (11). Finally, leptin-deficient mice display an increased sensitivity to TNF- and LPS-induced lethality, indicating that functional leptin is important in inflammation and its control (30, 33).
Cytokines have been implicated in the low to moderate inflammation associated with obesity and insulin resistance (34). In what appears to be a paradox, our patients demonstrated augmentation in cytokine release in the face of progressive insulin sensitivity. Thus, in all human studies to date, leptin deficiency has been associated with metabolic and endocrine pathology and possibly with parameters of immune responsiveness. Leptin replacement to the approximate physiological range has ameliorated these parameters, whereas high pharmacological or high physiological levels as seen in obesity do not appear to be linked to pathological or regulatory changes.
Conclusions
We evaluated the lymphocyte subset populations and in vitro responsiveness of PBMC isolated from a group of patients with leptin deficiency before and after leptin replacement as well as normal cells in the presence of leptin and other cytokines. The primary abnormalities seen were lower T cell subsets that corrected with leptin replacement. Additionally, TNF- production from patient cells was reduced at baseline and normalized with leptin replacement. It is important to note that the degree of TNF- production after r-metHuLeptin therapy did not exceed the normal range. Proliferation response and basal and PHA-stimulated IFN- production were unchanged before and after r-metHuLeptin therapy and similar to healthy controls.
Leptin has been implicated as an important immune modulator in various animal and human models. Although our patients with profound leptin deficiency had reductions of various T cell and cytokine levels at baseline, they demonstrated no evidence of immune dysfunction. Leptin replacement led to a correction of metabolic and hormonal abnormalities in the face of increasing T cell and cytokine concentrations, again without any phenotypic alterations in immune function. This provides additional evidence that the human situation is complex and that rodent models of general immune function and especially autoimmunity are of questionable relevance to the human condition. Furthermore, it demonstrates the need for continued research to elucidate the relative roles of leptin in modulating immune function.
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Footnotes |
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First Published Online November 29, 2005
Abbreviations: HbA1c, Glycosylated hemoglobin; IFN, interferon; LPS, lipopolysaccharide; NK, natural killer; PBMC, peripheral blood mononuclear cell(s); PHA, phytohemagglutinin; r-metHuLeptin, recombinant methionyl human leptin.
Received May 31, 2005.
Accepted November 17, 2005.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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receptor levels in humans with relative leptin deficiency. J Clin Endocrinol Metab 90:1625–1631, interleukin-12, and tumor necrosis factor- in human interferon- receptor 1 deficiency. J Infect Dis 178:1095–1104[Medline]This article has been cited by other articles:
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  J. Y. Park, A. Y. Chong, E. K. Cochran, D. E. Kleiner, M. J. Haller, D. A. Schatz, and P. Gorden Type 1 Diabetes Associated with Acquired Generalized Lipodystrophy and Insulin Resistance: The Effect of Long-Term Leptin Therapy J. Clin. Endocrinol. Metab., January 1, 2008; 93(1): 26 - 31. [Abstract] [Full Text] [PDF]
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, P < 0.05 compared with baseline.