Maristella A. Landgraf

Leptin Downregulates LPS-Induced Lung Injury: Role of Corticosterone and Insulin

Background/Aims: We investigated the effects of leptin in the development of lipopolysaccharide (LPS)-induced acute lung inflammation (ALI) in lean mice. Methods: Mice were administered leptin (1.0µg/g) or leptin (1.0µg/g) followed by LPS (1.5µg/g) intranasally. Additionally, some animals were given LPS (1.5µg/g) or saline intranasally alone, as a control. Tissue samples and fluids were collected six hours after instillation. Results: We demonstrated that leptin alone did not induce any injury. Local LPS exposure resulted in significant acute lung inflammation, characterized by a substantial increase in total cells, mainly neutrophils, in bronchoalveolar lavages (BAL). We also observed a significant lymphocyte influx into the lungs associated with enhanced lung expression of chemokines and cytokines (KC, RANTES, TNF-α, IFN-γ, GM-CSF and VEGF). LPS-induced ALI was characterized by the enhanced expression of ICAM-1 and iNOS in the lungs. Mice that received LPS showed an increase in insulin levels. Leptin, when administered prior to LPS instillation, abolished all of these effects. LPS induced an increase in corticosterone levels, and leptin potentiated this event. Conclusion: These data suggest that exogenous leptin may promote protection during sepsis, and downregulation of the insulin levels and upregulation of corticosterone may be important mechanisms in the amelioration of LPS-induced ALI.

Inflammatory milieu as an early marker of kidney injury in offspring rats from diabetic mothers.

The present study investigated the early presence of inflammatory response in renal tissue of young offspring from diabetic mothers. The effect of L-arginine (L-arg) supplementation was also investigated. The offspring was divided into four groups: group CO (controls); group DO (diabetic offspring); group CA (CO receiving 2% L-arg solution) and group DA (DO receiving the 2% L-arg solution). Glycemia, arterial pressure and renal function were evaluated; gene and protein expression of pro-inflammatory cytokines were also measured. Blood pressure levels were significantly increased in 2 and 6 month-old DO rats, whereas L-arg administration caused a significant decrease in the DA group, at both ages. DO rats showed a significantly blunted glycemic response to exogenous insulin. In 2 month-old DO animals, renal protein expression of pro-inflammatory molecules was significantly increased. At six months of age, we also observed an increase in gene expression of pro-inflammatory molecules, whereas L-arg supplementation prevented this increase at both ages. Our data suggest that activation of inflammatory pathways is present early in the kidney of DO rats, and that L-arg can attenuate the expression of these markers of tissue inflammation. Our results also reinforce the concept that intrauterine environmental factors are a fundamental determinant in the development of metabolic and vascular diseases later in life.

Generation and characterization of a spontaneously immortalized endothelial cell line from mice microcirculation

Endothelial cells from microvasculature are directly involved in a large number of vascular diseases; however, culture of these cells is problematic, since most methodologies employ proteolytic enzymes or mechanical techniques, leading to cell damage and contamination of endothelial cultures with other cellular types. Besides, primary cultured cells have a short life span in vitro and undergo replicative senescence after 3-4 passages, limiting long-term studies. In the present work we report the generation of a spontaneously immortalized endothelial culture obtained from mice pulmonary capillaries. Firstly, primary (third passage) and immortalized (100th) cultures were established. Further, monoclonal populations were obtained by serial dilutions from immortalized cultures. Cells were analyzed according to: (1) morphological appearance, (2) expression of specific endothelial markers by fluorescent staining [von Willebrand Factor (vWF), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE) and Ulex europaeus (UEA-1)] and by flow cytometry (endoglin, VE-cadherin and VCAM-1), and (3) release of nitric oxide (NO), assessed by the specific fluorescent dye DAF-2 DA, and prostacyclin (PGI2), quantified by enzyme immune assay. In both cultures cells grew in monolayers and presented cobblestone appearance at confluence. Positive staining for vWF, eNOS, ACE and UEA-1 was detected in cloned as well as in early-passage cultured cells. Similarly, cultures presented equal expressions of endoglin, VE-cadherin and VCAM-1. Values of NO and PGI2 levels did not differ between cultures. From these results we confirm that the described spontaneously immortalized endothelial cell line is capable of unlimited growth and retains typical morphological and functional properties exhibited by primary cultured cells. Therefore, the endothelial cell line described in the present study can become a suitable tool in the field of endothelium research and can be useful for the investigation of production of endothelial mediators, angiogenesis and inflammation.

Modulation of Lung Allergic Inflammation and Malnutrition

Nutritional deficiency is commonly associated with a significantly impaired immune response, particularly in relation to cell-mediated immunity, the complement system, cytokine production and phagocyte function. However, there are few data on the consequences of nutritional deficiency in allergic diseases of the lung. In fact, malnutrition is the most common cause of immunodeficiency worldwide. Several studies have indicated that the incidence of alterations in lung functions can be associated with birth weight, specifically with maternal malnutrition, but data linking intrauterine undernutrition with allergic diseases of the lung are lacking. The purpose of this review is to associate malnutrition, including intrauterine malnutrition, with the establishment of immune responses and the development of lung allergic inflammation.

Modulation of Lung Allergic Response by Renal Ischemia and Reperfusion Injury

The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). In addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. In addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors.

Reduction in Inflammatory Markers Expression in Serum is Related to Improvement in Renal Functions in Intrauterine Undernourished Rats

Background and aims: Maternal undernutrition can induce a range of fetal adaptations, which can lead to permanent alterations in adulthood. Interleukin (IL)-18 play an integral role in tubular injury and the development of renal dysfunction during a variety of inflammatory processes. In this work, we have investigated the impact of intrauterine undernutrition on the inflammatory markers, and the correlation of these markers with the renal dysfunction; the effect of L-arginine (L-arg)administration on those parameters also was investigated.Methods: Female Wistar rats were randomly divided into 2 groups: nourished(NR-ad libitum diet) and undernourished (UR-50% food restriction). After birth, each litter was left with the mother for 28 days. Some of the offspring received a 2% L-Arg solution in drinking water (groups NR+L-Arg and UR+L-Arg). Proteinuria and glomerular filtration rate (GFR) were determined. IL-18 and cytokine induced neutrophil chemoattractant-2 (CINC-2) were evaluated by Bioplex, in serum.Results: In UR, the proteinuria (63%), IL-18 (121%) and CINC-2 (61%) were increased, when compared to NR, and the L-arg treatment abolished these effects; the GFR was decreased in UR (43%), and the L-arg treatment prevented this reduction.Conclusions: In UR, the inflammatory markers, such as IL-18, are present in an early stage after birth, probably contributing for the development of renal injury in this model. The lower expression of these markers seems to be directly related to improvement of renal function. Our results suggest that these inflammatory markers can be attenuated by L-arginine.Supported by FAPESP, CNPq, and INCT Complex Fluids

Role of Maternal and Infant Malnutrition on the Development of the Inflammatory Response

The thrifty phenotype hypothesis holds that intrauterine malnutrition leads to an adaptive response that alters the fetal metabolic and hormonal milieu designed for intrauterine survival. This fetal programming predisposes individuals to several diseases in adulthood. Fetal nutrition is an important key regulator of fetal growth and thus an obvious candidate as a possible programming influence. Fetal overexposure to maternal glucocorticoids triggers programming events in utero; these effects appear to be relevant to changes in utero because there are strong correlations between birth weight, plasma cortisol concentrations, and the development of hypertension and type 2 diabetes. Malnutrition, an important cause of immunosuppression and undernutrition during critical periods of gestation, neonatal maturation, and weaning, can lead to clinically significant immune deficiency and infections in children. Micronutrients have a relationship to antibody formation and the development of the immune system, and micronutrient deficiencies are related to poor growth, impaired intellect, and increased mortality, and susceptibility to infection.

A comparative study of pathophysiological alterations in scorpionism induced by Tityus serrulatus and Tityus bahiensis venoms

ABSTRACT Scorpionism is a relevant public health problem in several countries in tropical and subtropical regions. In Brazil, Tityus serrulatus sting can induce acute lung injury in part as a consequence of inflammation. Despite the occurrence of other scorpions of Tityus genus in Brazilian scorpiofauna, the knowledge regarding pulmonary alterations is related to T. serrulatus venom (Tsv). Here we studied, comparatively, the pathophysiological changes in the rat airways envenomed by Tsv or T. bahiensis venom (Tbv), since both scorpions are involved in human accidents but with severe envenomations occurring when victims are stung by T. serrulatus. After intravenous injection of the venoms (200 &mgr;g/kg), both were able to induce Evans blue extravasation (in 30 min) into airways and increased protein extravasation into lungs at 4 and 24 h, but the magnitude of such events was higher in Tsv group. Hemorrhage (in 60 min) in the lungs was higher in Tbv group, while IL‐1&bgr; (at 1 h) and IL‐6 (at 1 and 4 h) in lung homogenates were detected only in Tsv group. Four and 24 h after envenomation, myeloperoxidase activity in lung was equally augmented in the envenomed groups, as well as an increased in polymorphonuclear cell numbers in bronchoalveolar lavage fluid. At 4 h blood leukogram showed increased leukocyte values with the highest neutrophilia in Tsv group. The numbers of leukocytes and neutrophils remained higher than control at 24 h in Tsv and Tbv groups, and it was accompanied by lympho (envenomed groups) and monocytosis (Tsv group). In conclusion, although Tbv was capable of inducing acute lung injury and blood leukocyte mobilization, most of the evaluated parameters were more affected by the Tsv. These results could help to explain the pathophysiology of the scorpionism and the clinical data arguing toward the greatest severity associated with T. serrulatus stings. HighlightsT. bahiensis venom also induced pathophysiological alterations in rat airways.Lung edema was higher in T. serrulatus envenomed rats.Cytokines were found only in the lungs of rats envenomed by T. serrulatus venom.Leukocyte mobilization to lungs was similar in envenomed rats.Different venoms toxins and mediators in the envenomation may explain our findings.

Intrauterine Malnutrition Reduced Long Leptin Receptor Isoform Expression and Proinflammatory Cytokine Production in Male Rat Pulmonary Endothelial Cells Stimulated by Lipopolysaccharide

Background/Aims We have previously shown that low birth weight (LBW) rats exposed to intrauterine malnutrition have an impaired lung inflammatory response and reduced levels of inflammatory mediators; however, circulating leptin levels were not increased. We evaluated long leptin receptor isoform (ObRb) expression in lung endothelial cells from low birth weight rats and examined its role in the production of lipid mediators and cytokines. Methods Lung endothelial cells were obtained from normal birth weight (NBW) rats or LBW rats subjected to intrauterine malnutrition. These cells were stimulated with leptin (10 ng/mL), LPS (lipopolysaccharide, 1 μg/mL), or leptin plus LPS. Six hours after stimulation, the production of inflammatory mediators (PGE2, LTB4, IL-1β, and IL-6) was evaluated using commercial ELISA kits, and Western blotting was performed to investigate p38MAPK, NF-κB, and ObRb expression. Results Leptin increased IL-1β levels in only cells from the NBW group, whereas LPS increased PGE2 and LTB4 levels in cells from both groups; leptin addition potentiated lipid mediator production induced by LPS in the NBW group. LPS enhanced the production of IL-1β and IL-6 in only endothelial cells from NBW rats. Leptin receptor expression was decreased (63%) in endothelial cells from LBW rats. None of the stimuli increased NF-κB or p38 signaling pathway expression in cells from LBW rats. Conclusion These results suggest that intrauterine malnutrition compromises leptin receptor expression and cytokine production in pulmonary endothelial cells stimulated by LPS; these effects seem to involve the NF-κB and p38MAPK signaling pathways.

The Influence of Maternal Diabetes on the Expression of Inflammatory Markers in the Offspring

Background and aims: Previous studies from our group have shown that maternal diabetes is an important cause of glomerular hypertrophy in the offpring (DO) and that hypertension linked to an impaiment in the vascular endothelium-dependent relaxation is early detected. The present study was designed to evaluate the expression of cytokines/chemokines in plasma and renal tissue from DO and the possible effect of L-arginine administration on those parameters.Methods: Diabetes mellitus was induced in Wistar female rats with a single dose of streptozotocyn, 50mg/kg, IP. Diabetic dams were caged overnight with a male. After birth, each litter was left with the mother for 28 days. Some of the offspring received a 2% L-Arg solution in drinking water (groups DA and CA, controls+ L-arg). Tumor necrosis factor-alpha (TNF-α), interleukin (IL) -6 and IL-1b were measured by real time PCR in the kidney from 2 and 6 month-old rats.Results: Serum expression of TNF-α, interleukin (IL)-1b, IL-2, IL-17, interferon (IFN)-g, macrophage inflammatory protein (MIP)-1and leptin enhanced in DO group and L-arg prevented this increase. TNF-α, interleukin (IL) -6 and IL-1b (measured by real time PCR) in the kidney from DO, 6 month-old, were also increased but returned to control values in DA.Conclusions: Renal and serum inflammatory pathways seem to be early activated in DO, contributing or being the triggering factor for the development of hypertension and renal injury in this model. Our results suggest that these inflammatory pathways can be attenuated by L-arginine.Supported by FAPESP and CNPq