Expression of resistin , CXCR 3 , IP-10 , CCR 5 and MIP-1 α in obese patients with different severity of asthma

Asthma studies suggest that alteration in the infl ammation pattern may be associated with the severity of asthma. The aim of this study was to compare in vitro the expression of chemokines, chemokine receptors and cytokine production from CD4+ T human lymphocytes of asthmatic, both obese and non-obese patients with diff erent severity levels of asthma. Lymphocytes were labeled with monoclonal anti-human CXCR3/IP-10, MIP-1α/CCR5 antibodies and were analyzed by fl ow cytometry. Cell culture supernatants were used to measure production of interleukin IL-6 and resistin by ELISA. CXCR3/IP-10 expression increased in non-obese patients with mild persistent asthma (2.2%, p<0.05), moderate persistent asthma (3%, p<0.003) and severe persistent asthma (4%, p<0.004); this eff ect was stronger in obese patients with severe persistent asthma (35%, p<0.004). MIP-1 α / CCR5 increased in non-obese patients with intermittent asthma (0.65%, p<0.05) and severe asthma (1.4%, p<0.03); in obese patients, this expression was greater in intermittent asthma (8%, p<0.05) and severe persistent asthma (12%, p<0.04). Resistin production strongly increased in obese patients with intermittent (976 ng/ml) and severe persistent asthma (795 ng/ml). IL-6 increased in both lean and obese persons; however, the highest value was registered in the group of severe persistent obese asthmatics (992 pg/ml). Obesity per se increased the infl ammatory profi le of chemokines / cytokines secreted by cells of the blood, increasing the infl ammatory status in asthmatic patients. Resistin showed characteristics of a pro-infl ammatory cytokine mainly in severely obese asthmatics.

levels of resistin and the body mass index (BMI) and its role in insulin sensitivity (Bastard et al., 2002;Fasshauer and Paschke, 2003).However, some studies have reported increased resistin levels in overweight subjects and type 2 diabetes (McTernan et al., 2002;Vidal-Puig and Orahilly, 2001).Resistance to a cortisone-based treatment seems to be a failure in inhibiting cytokine production by lymphocytes, suggesting that diff erent infl ammatory cytokines which are associated with chemokines and chemokine receptors in the airways may be present in patients with diff erent degrees of severity of asthma (Shannon et al., 2008).Asthma and obesity are public health concerns that have high impact and an increasing prevalence in recent years (Shore, 2007(Shore, , 2008)).Prospective studies have demonstrated that obesity is a risk factor for the development and exacerbation of asthma, and a positive correlation between body mass index and the development of asthma has been observed (Segura et al., 2007).
Other studies have shown that severe asthma has a higher prevalence in overweight patients compared to normal weight patients and that body mass index (BMI) is positively associated with clinical asthma severity.In addition, patients with more severe asthma have higher BMI than those with milder asthma (Saint-Pierre et al., 2006).Obesity has mechanical eff ects on lung function; it leads to a systemic proinfl ammatory status, thereby potentially increasing airway infl ammation, and is associated with a number of co-morbid factors which might interfere with asthma control.Obesity does not cause airfl ow obstruction, but can result in pulmonary restriction and reduction in airway diameter, which could contribute to airway hyper-responsiveness. Mouse asthma models have demonstrated that obesity and cytokines can enhance airway hyper-responsiveness, airway infl ammation and allergic responses, but it is unclear whether obesityassociated infl ammatory mechanisms are relevant in human asthma (Beuther, 2009).The aim of this study was to compare, in vitro, the expression of chemokines, chemokine receptors and cytokine production by peripheral blood lymphocytes from healthy subjects and patients with diff erent severity of asthma, with or without obesity.

Subjects
All patients and volunteers gave their written informed consent for interviewing and blood sampling.The study was conducted according to the recommendations of the Declaration of Helsinki and approved by the local Ethics Committee.The sample consisted of fi fty asthmatic patients, diagnosed with allergic asthma according to The Global Initiative for Asthma (GINA, 2008), which establishes a practical system of classification considering clinical and functional aspects such as frequency of diurnal and nocturnal respiratory symptoms and lung function; their combination allows us to classify asthma severity as: a) intermittent asthma, in which the main symptoms of breathlessness of varying intensity and duration, bronchial spasms, cough, mucus secretions, and wheezing occur less than once a week, maximum volume exhaled in the first second of a forced expiration (FEV 1 ) equal to or greater than 80% of predicted with less than 20% variability, b) mild persistent asthma, which consists of symptoms that occur at night, more than once a week, FEV 1 greater than or equal to 80% of predicted with lower variability of 20-30%, c) moderate persistent asthma, with exacerbated symptoms that aff ect activity and sleep and with the use of short-acting β2 agonists on a daily application, FEV 1 from 60 to 80% of predicted with greater than 30% variability, and d) severe persistent asthma, with daytime and nighttime symptoms that are frequently exacerbated and the use of short-acting β2 agonists with daily application, FEV 1 less than or equal to 60% of predicted with greater than 30% variability.We included clinical symptoms, spirometry and skin prick tests.In addition, we applied the following criteria: a) inclusion; age between 18 and 65 years for both sexes, obese and non obese (14 obese men and 12 women, 10 non-obese men and 14 women, 12 healthy subjects), residents of the metropolitan area of Mexico City, b) exclusion; patients with pulmonary diagnosis other than allergic asthma, history of immunotherapy in the past year, history of immunodefi ciency or autoimmune diseases, smoking history and / or patients who have cooked with wood, confi rmed pregnancy, who have used topical or systemic antihistamine-based therapies within two weeks prior to entering the study , who have received astemisol within the last two months prior to the execution of the skin prick test for mites, the presence of hives or dermographism, asthma attacks at any stage and residence less than a year in Mexico City.Patients were divided into three groups according to their body mass index (BMI) following the guidelines established by the World Health Organization (WHO) (Pauwels et al., 2001): obesity (BMI ≥ 30 kg/m 2 ), and non-obese (BMI < 30 kg/m 2 ) and healthy.After the taking the blood sample, the patients were treated with a combination therapy based on mometasone / formoterol (260 μg) (Merck, California., USA) and oriented to control environmental factors (dust, pollen, cigarette smoke etc) that trigger asthma exacerbation.Healthy non-smoking subjects without obesity, hypertension, diabetes, or any other metabolic symptoms were used as controls.

Isolation of mononuclear cells from healthy subjects and patients with asthma
Fifteen ml of venous heparinized blood were obtained from asthmatic patients, both obese and non-obese, and healthy non-smoking adult volunteer donors (n= 62) of both sexes.The blood was diluted 1:3 with phosphate buff ered saline (PBS; 0.15 M phosphate buff er).Blood was layered over 3 ml of Ficoll-Hypaque (Sigma Chemical Co., St. Louis, Missouri, USA) with gradient (δ = 1.077) and centrifuged at 1500 rpm for 30 min at 21 °C (Böyum, 1968).The cellular interface peripheral blood mononuclear cells (PBMC) were collected and washed twice with PBS.Cell viability was determined using Trypan blue dye (Sigma Chemical Co., St Louis, MO); exclusion was ≥ 90%.

Cell Purifi cation
The cellular interface peripheral blood mononuclear cells (PBMC) were removed and washed two times with PBS.CD4+ T cells were purifi ed using the CD4+ T cell isolation kit II, as an indirect magnetic labeling system for the isolation of untouched CD4+ T cells from human PBMCs (Miltenyi Biotec, Germany), and LS column and MidiMACS separator.Briefl y, 1 x 10 7 PBMC cells were placed in propylene tubes with 80 μl PBS-albumin-EDTA and 20 μl cocktail of biotin-conjugated antibodies against CD8, CD11b, CD16, CD19, CD36, CD56, CD123, TCRγ/δ and CD235a (Glycophorin A) and were incubated for 10 min at 4 °C.These cells were subsequently labeled magnetically with Anti-Biotin MicroBeads for depletion.The CD4+ lymphocytes obtained were 95% pure.Acquisition of 10,000 events was conducted in fl ow cytometry FACSaria (BD Biosciences, Palo Alto, CA).For analysis Cell Quest software, version 3.1 (Becton Dickson, San Jose, CA) was used.
Briefl y, 5 X 10 5 CD4+ T cells from each group were incubated in 24-well plates for 24 h; after incubation, cells were centrifuged for 5 min at 400 g and supernatants were aspirated without disturbing the pellets.Cells were washed with PBS/0.5% albumin/2mM EDTA.They were then marked with mAb and incubated for 20 min at 4 °C in the dark, and fi xed with 1% p-formaldehyde (Sigma Chemical Co., St. Louis, MO) according to the manufacturer's instructions (PharMingen).Acquisition of 10,000 events was conducted in fl ow cytometry FACSaria (BD).For analysis, FACS Diva 6.1 software was used Miltenyi, et al., 1990.

Quantifi cation of resistin and IL-6
Resistin and IL-6 in the cell culture supernatants were measured using commercially available enzyme-linked immunosorbent assay kits (ELISA) (Peprotech®, Rocky Hill, NJ).

Statistical analysis
The statistical analysis was performed using the SPSS® software, version 14.0 (SPSS Inc., Chicago.Illinois, USA) and MS Excel 2007.Quantitative data was expressed as mean and standard deviation (SD).Statistical comparison among groups was performed using the Mann-Whitney U tests.Diff erences were considered as statistically signifi cant when p<0.05.The relationship between in resistin production and the in IL-6 production were analyzed using normal distribution histograms with confi dence intervals of 95%.

CXCR3 and IP-10 expression
CXCR3 and IP-10 expressions were compared among obese and non-obese patients with with diff erent degrees of severity of asthma (intermittent, mild persistent, moderate persistent, and severe persistent asthma), as well as with healthy subjects.In non-obese patients, CXCR3 and IP-10 expressions were not signifi cantly diff erent in intermittent asthmatic compared to controls (healthy subjects); this expression, however, increased gradually in patients with mild persistent asthma, moderate persistent asthma and severe persistent asthma, with statistically signifi cant diff erences among groups (2.2%, 3% and 4% respectively; p< 0.004 ) (Fig. 1a).In obese patients with intermittent asthma, mild persistent asthma, moderate persistent asthma and severe persistent asthma CXCR3 and IP-10 were signifi cantly over-expressed (19%, 16%, 13% and 35% respectively; p<0.04) compared to healthy subjects and non-obese patients (Fig. 1b).Meanwhile, the expression was signifi cantly lower in moderate persistent obese asthmatic (13%, p< 0.05) and was the highest for obese patients with severe asthma (35%, p<0.001) (Fig. 1b).

MIP-1α and CCR5 expression
The expression of MIP-1α and CCR5 in non-obese asthmatic patients was significantly increased in patients with intermittent asthma and severe persistent asthma (0.6% and 1.4%, p< 0.05 and p< 0.03 respectively) compared to healthy subjects.Expression levels in mild persistent asthmatic and moderate persistent asthmatic were lowest (0.3% and 0.4%, p<0.04 and p<0.05 respectively) as shown in Figure 2a.MIP-1α and CCR5 expressions in obese, asthmatic patients increased signifi cantly (8%, 6%, 7% and 12%, p< 0.04 respectively) in all groups compared to healthy subjects (Fig. 2b).There were considerable diff erences in the severe persistent asthmatic group (12%, p< 0.04) compared to other groups (Fig. 2b).We also observed over-expression of MIP-1α and CCR5 in obese patients compared to non-obese patients.

Production of cytokines IL-6 and resistin in obese and non-obese asthmatics patients compared to healthy subjects
The concentration of resistin strongly increased in obese patients with intermittent asthma and severe persistent asthma (976 ng/ml and 795 ng/ml, respectively) (Fig. 3).Therefore, there was an increase in the production of resistin in obese patients compared to lean patients: Intermittent 976 vs. 190 ng/ml, moderate persistent 480 vs. 378 ng/ml and severe persistent 795 vs. 612 ng/ml; except for the lean subjects with mild persistent asthma 715 vs. 417 ng/ml as shown in Figure 3.All obese patients, regardless of the degree of their disease, showed high levels of IL-6 compared to lean patients: intermittent asthma (742 vs. 600 pg/ml), mild persistent asthma (776 vs. 552 pg/ml), moderate persistent asthma (902 vs. 712 pg/ml) and severe persistent asthma (992 vs. 839 pg/ ml) (Fig. 3).

DISCUSSION
The coordinated expression and binding of chemokines to their receptors seem to be diff erentially regulated in specifi c stages of asthma.In this study, CXCR3/ IP-10 and CCR5/ MIP-1α were overexpressed in obese patients with severe persistent asthma, which is in agreement with the reported of Brasier (2008) in patients with severe persistent asthma who underwent bronchoalveolar lavage.
We also showed that obesity per se increased the infl ammatory status of asthmatic patients, probably due to the chemotactic eff ect of chemokines secreted by the blood cells of asthmatics.Additionally, cell activation may modify the expression of chemokines and chemokine receptors, which, in alternate fashion, are essential for leukocyte recruitment during infl ammation.Once activated, T lymphocytes acquire diff erent migratory capacities and are, in fact, the key factor for an effi cient immune-response regulation.Interestingly, we found an association between increased expression of CXCR3 / IP-10 and severity of disease in non-obese asthmatic patients, while in obese patients increased expression of these markers was only observed in intermittent obese asthmatics and obese individuals with severe persistent asthma.The classifi cation of asthma severity was very useful to us to relate the diff erent levels of asthma severity to the expression of infl ammatory markers studied; however, asthma, as a chronic infl ammatory disease shows variations in clinical manifestations and the degree of airfl ow obstruction, so its severity may change over time in the same patient, indicating that not all infl ammatory markers correlate with levels severity of asthma, as reported in the intermittent asthma group, especially in obese patients.
The elevated expression of these molecules in humans in primary and more severe stages of the disease had not been previously reported.We show that higher BMI scores  b) obese patients with intermittent, mild persistent, moderate persistent and severe persistent asthma and healthy subjects.The cells were isolated by the method of Böyum and purifi ed to CD4 + T cells by the negative selection technique and were cultured for 24 h.They were stained with fl uorochromo-conjugated (FITC or PE) anti human IP-10 and CXCR3 monoclonal antibodies.10,000 events were analyzed by fl ow cytometry.The quadrants were set with the isotype control (FITC or PE-conjugated mouse IgG).Bold histograms and numbers in the dot plots represent the mean ± SD of positively stained cells from at least six independent experiments.The statistical differences between the groups were calculated using the Mann-Whitney U test.p <0.05 was considered signifi cant.

Fig 2: MIP1 α and CCR5 expression in lymphocytes from peripheral blood
Five x 10 5 CD4+ lymphocytes were obtained from non-obese and obese patients with intermittent, mild persistent, moderate persistent and severe persistent asthma and healthy subjects and were cultured in 24-well plates in a humidifi ed chamber at 37 °C and 5% CO 2 for 24 h.Cells were stained with fl uorochromo-conjugated (FITC or PE) anti human MIP 1-α and CCR5 monoclonal antibodies.At least 10,000 events were analyzed by fl ow cytometry.Black bars and bold numbers in the dot plots represent the mean ± SD from fi ve independent experiments.The quadrants were set with mouse IgG isotype control.The statistical differences between the groups were calculated using the Mann-Whitney U test (p <0.05).
and elevated CXCR3/IP-10 expression levels (up to 9 times) are associated with increased asthma severity added to the condition of obesity.These fi ndings highlight the importance of controlling theinfl ammation taking into account the role of obesity and it provides support for the assumptions that obesity-induced inflammation may contribute to greater asthma severity.The strong correlation between CXCR3 expression and Th1 diff erentiation led us to the hypothesis, subsequently verifi ed in mouse models, that CXCR3 and its ligands regulate the migration of Th1 cells into sites of Th1driven infl ammation (Lin et al., 2011); it also correlates with a study in which the expression of CXCR3 and its ligand IP-10 (CXCL10) was induced by IFN-γ in mice challenged with an allergen in the lung (Gangur et al., 1998).High levels of IP-10 and CXCR3 have been reported in co-cultured cells in the presence of INF-γ in patients with chronic obstructive disease.IP-10 diff ers from other chemokines in its apparent specifi city to activate T lymphocytes.We believe that it is also relevant in regulating the production and synthesis pattern of cytokines.These data imply that CCR5 and CXCR3 could be a potent therapeutic target of asthma for inhibiting the recruitment of T-cells to the airways.
Some limitations of the study deserve further discussion.To identify cells that express on their cell surface characteristic infl ammatory markers we have often used invasive techniques such as bronchoalveolar lavage fl uid, which was not performed in this study.Based upon the principle that cells migrate selectively to the infl ammatory focus through chemokines and their receptors, a peripheral blood sample is less painful for the patient.In addition, the chemokine system in vivo is extremely redundant due to the large number of diff erent chemokines, the overlap in chemokine function and the pleiotrophy of chemokine-receptor interaction.One of the challenges in this fi eld is to identify which receptors play major roles in specifi c infl ammatory conditions.The current fi ndings suggest that the Th2-linked (CC) receptors are not the only potential targets for asthma therapy.The Th1-linked (CC) receptors, especially CCR5 and CXCR3, may also be valid targets.It was demonstrated that these markers play an important role in migratory behavior of peripheral blood T cells and infl uence the activation of T helper cells; however, we must be cautious in extrapolating these results to other population of lymphocytes such as CD8+ T cells and regulatory T cells.
The levels of many cytokines and other mediators may be elevated in obese patients and many be involved in systemic infl ammation, such as interleukin 6, TNF-α, and reactive C protein.Our data showed high concentrations of resistin and IL-6 in overweight asthmatic patients compared to healthy individuals, consistent with what has been reported in the literature (Bokarewa et al., 2005).In a previous study it was demonstrated that an increase in resistin is associated with the severity of asthma, which results in an increased expression of adhesion molecules such as VCAM-1 and ICAM-1, as well as pro-infl ammatory cytokines IL-6 and TNF-α, which trigger damage (Mondragón-González et al., 2007).IL-6 also activates β1-integrin, which is a component of the VLA-4 complex and the ligand for fi bronectin and VCAM-1, and thereby could infl uence cell attachment (Clahsen and Schaper, 2008).In this study, a strong increase in resistin and IL-6 levels was observed in obese patients with severe persistent asthma, while in thin patients with the same disease the production of these cytokines was signifi cantly lower.Generally in overweight or obese individuals a considerable increase in adipocytes occurs, which increases IL-6 and resistin levels, and these in turn activate endothelial cells to stimulate migration of leukocytes and accumulation of leukocytes in the fatty adipose tissue.Under these conditions the increase in fatty tissue and expression of infl ammatory cytokines contribute to the chronic infl ammatory status, and they are characteristics of obesity.We observed this phenotype in intermittent asthma and severe asthma patients compared to the other asthma groups and healthy subjects.The production level of resistin in obese intermittent patients was elevated.This may be because resistin behaves as a prototype infl ammatory cytokine, favoring the infl ammatory process, and it also may have an additive eff ect with the infl ammation that aff ects obesity.In lean patients the increased production of resistin was observed in patients with moderate persistent asthma and severe persistent asthma; of severity exacerbation of clinical symptoms was present at these levels.The relation between high concentrations of resistin and asthma severity obtained in our study agrees with the results obtained in in vivo studies (Mishra et al., 2007), in which it was found that resistin and IL-13 cause infl ammation in airways and pulmonary remodeling in the lung of mice, resulting in the deposit of perivascular and peribronchial collagen fi bers.Additionally, IL-6 and resistin could be induced by diff erent allergens independent of Th2-cells, with biological and clinical adverse eff ects in patients with exacerbated asthma (Wolpe et al., 1998).In obese patients there is a condition of chronic systemic infl ammation, which also means that if they have asthma, it is more severe than in lean persons and is diffi cult to control.In this study we found that the greater the overlap of groups, for example in severe persistent asthmatic obese it was poorer its fi nal clinical status.In addition, obese asthmatics, regardless of the degree of severity of the disease showed high levels of IL-6, compared to healthy and non-obese patients.Hence there is a direct relationship between obesity, production of IL-6 and resistin, and cells migrating from the bloodstream the to infl ammatory focus.We have previously reported that asthmatic patients with a BMI ≥ 30 had a high concentration of leptin, especially women.In the current study, we showed that concentrations of IL-6 and resistin, together with obesity, can be considered as indicators of asthma severity.

CONCLUSION:
Obesity per se increased the inflammatory profile of chemokines / cytokines secreted by cells of the blood by increasing the infl ammatory status of asthma.Resistin shows characteristics of a pro-inflammatory cytokine, mainly in severely obese asthmatics.Chemokine receptors and their ligands, such as CXCR3/IP-10 and CCR5/MIP-1α, should be considered key infl ammatory molecules in obese patients with diff erent levels of asthma severity.

Fig 1 :
Fig 1:Expression of IP-10 and CXCR3 in lymphocyte populations obtained from healthy subjects, non-obese patients with asthma and obese patients with asthma Five x 10 5 CD4+ lymphocytes were obtained from human peripheral blood (a) non-obese and (b) obese patients with intermittent, mild persistent, moderate persistent and severe persistent asthma and healthy subjects.The cells were isolated by the method of Böyum and purifi ed to CD4 + T cells by the negative selection technique and were cultured for 24 h.They were stained with fl uorochromo-conjugated (FITC or PE) anti human IP-10 and CXCR3 monoclonal antibodies.10,000 events were analyzed by fl ow cytometry.The quadrants were set with the isotype control (FITC or PE-conjugated mouse IgG).Bold histograms and numbers in the dot plots represent the mean ± SD of positively stained cells from at least six independent experiments.The statistical differences between the groups were calculated using the Mann-Whitney U test.p <0.05 was considered signifi cant.

Fig 3 :
Fig 3: Resistin and IL-6 production by lymphocyte cells obtained from healthy subjects, lean patients and obese patients with different levels of severity of asthma Cell culture supernatants were analyzed by ELISA to determine IL-6 and resistin production.The bars represent the average of three independent experiments in each group and the healthy subjects (control group).Quantitative data were expressed as normal distribution histograms with confi dence intervals of 95%.