IL-1 β , IL-6 And TNF-Alpha Secretion by PBMNC Stimulated with Polyclonal Antibodies Anti-Hrage ( AGE-Like ) is Enhanced by Hyperglycemia in Diabetes

Receptor for advanced glycation end products (RAGE) activation is known to play an important role in the development of diabetes complications by amplifying the inflammatory process. Herein, we examined the effect of polyclonal antibodies against human RAGE (Ab anti-hRAGE) on “primed” peripheral blood mononuclear cells (PBMNC by hyperglycemia in type 2 diabetes mellitus (T2DM) patients in comparison to healthy control. ROS generation and IL1β, IL-6 and TNF-α secretion were studied. PBMNCs were purified utilizing Ficoll-hypaque gradient. ROS was quantified by luminol-dependent chemiluminescence. Antibodies antihuman RAGE (AGE-like) was purched from Sigma Co. The cytokines were from PBMNC culture in presence or in the absence of anti-hRAGE. IL-1β, IL-6 and TNF-α were quantified in the supernatant of Ab anti-hRAGE-stimulated PBMNCs trough ELISA. Ab anti-hRAGE (AGE-like) significantly inhibited ROS production in unstimulated or stimulated PBMNCs from T2DM and healthy controls in a similar way. The percentage of inhibition was greater in primed PBMNC by hyperglycemia in diabetes (T2DM patients). In contrast, anti-hRAGE increased secretion of IL-1β, IL-6 and TNF-α (p < 0.05) in the supernatant of Ab anti-hRAGE-stimulated PBMNCs from T2DM patients compared with healthy controls (p < 0.05). The effect of antibody anti-hRAGE (AGE-like) in primed cells by hyperglycemia in diabetes may activates different signaling network when interact with RAGE on cells surface. Dual results induced by anti-hRAGE (AGE-like) associated with oxidizing response and pro-inflammatory cytokine secretion suggest activation of several signaling network in AGE-RAGE interaction. It may have consequences on innate immunity.


Preparation of peripheral blood mononuclear cells (PBMNC)
PBMNCs were purified from 10.0 mL of heparinized venous blood, using a Ficoll-Hypaque gradient as previously described [31], with slight modifications.The trypan blue exclusion test showed that the cell viability in all samples was of > 95%.

Determination of reactive oxygen species (ROS)
Modulations in the generation of ROS were estimated using the luminol quantitative chemiluminescence assay in a Magic Lite luminometer, (Ciba Corning Co., Medfield, MA, USA).The PBMNCs sample was washed in phosphate buffered saline (PBS) and a suspension containing 1 × 10 6 PBMNCs/mL PBS was transferred to an unsealed luminescence tube.Luminol (200 µL) dissolved in 0.4 M dimethyl sulphoxide was added to the sample, the final volume of the mixture was adjusted to 500 µL with PBS.The chemiluminescence [expressed in relative ligh units (RLU)/min] of each assay mixture was measured for 15 min (basal ROS production), following which addition of the Ab anti-hRAGE (100 ng/100 µL) to the reaction mixture and chemiluminescence measured for 40 min.Finally, NADPH oxidase inhibitor (DPI) (10 μM; 100 μL) was added to the reaction mixture and chemiluminescence measured for additional 15 min.The chemiluminescence was also made in the presence of PDB.In this case, basal ROS production was measured for 15 min, following which 20 μL of 10 −4 M PDB was added and ascendant ROS production was determined for 25 min; finally Ab anti-hRAGE (100 ng/100 µL) was added and chemiluminescence recorded for a further 20 min.

Statistical Analyses
The values are presented as the means ± standard deviation (SD).The nonparametric Kolmogorov-Smirnov test was used to assess the normal distribution of the continuous variables.Comparisons between groups were performed using unpaired Student's t-tests.In some experiments, we also used the chi-square test.All analyses were considered significant at p-values < 0.05 using Origin 6.0 (Microcal Software Inc., Northampton, MA, USA).

Suppression of ROS generation in PBMNCs from T2DM and healthy control by either antibody anti-hRAGE or NADPH-oxidase system inhibitior (DPI)
ROS, expressed as RLU/min, produced by unstimulated PBMNCs from T2DM patients produced higher levels of ROS (186.6 ± 39.0) compared to healthy controls (96.6 ± 11.0) (p < 0.05).The use of polyclonal antibodies against human RAGE suppressed ROS generation 29% and 19% in cells from T2DM and healthy controls, respectively (p > 0.05 by chi-square test).Similar suppression of ROS production was observed in PBMNCs assayed in the presence NADPH oxidase inhibitor (DPI).The percentages of inhibition were 48.0% for T2DM and 31.0%for healthy control (p > 0.05) (Table 2 and Figure 1).PBMNC from patient were more sensitive to inhibition than that from healthy control.

Antibody anti-hRAGE reduced ROS generation in PBMNCs PDB-stimulated from T2DM and healthy control
The role of Ab anti-hRAGE on ROS production by PBMNCs PDB-stimulated is shown in table 2 and figure 2. The phorbol ester activated PBMNCs-ROS derived from T2DM and healthy contols, the results, expressed as percentage of activation, were 166% and 67% respectively (p < 0.05).The PBMNCs-ROS derived PDB-stimulated was inhibited by Ab anti-hRAGE.The results expressed as percentage of inhibition were 55% and 53% for T2DM and healthy controls, respectively.However, the kinetics studies (Figure 1 and Figure 2) demonstrate that the effect of anti-

Discussion
The present study demonstrated that polyclonal antibodies against human RAGE (AGE-like) enhanced cytokines secretion and inhibited ROS generation in PBMNC from type 2 diabetic patients.It has been reported that the pathological consequences of interaction between RAGE with respective ligand leads to the generation of oxidative stress and upregulation of inflammatory pathways [9,[32][33][34].The blockade of RAGE with ligands has been suggested as a possible therapeutic target for controlling inflammatory process.In diabetic animal models, the blockade of RAGE, using soluble RAGE (sRAGE), demonstrated reduction in vascular inflammation and atherosclerotic lesion area and suppression in periodontitisassociated alveolar bone loss [10,[35][36][37].RAGE deficient animal models showed improvement of nephropathy, suppression of kidney injury and diabetes-accelerated atherosclerosis [38][39][40].Moreover, anti-RAGE antibodies increased survival in experimental models of severe sepsis, protected against AGE-mediated podocyte dysfunction and suppressed pro-inflammatory activities of human umbilical venular endothelial cells (HUVECs) induced by HMGB1 [41][42][43][44].Thus, theoretically, the use of antibodies anti-RAGE could downregulate inflammatory signaling pathways.
ROS overproduction hyperglycemia-induced result in oxidative stress and it plays a central role in the pathogenesis of diabetes complications [5,6].Activation of RAGE results in the generation of ROS in dependence of NADPH oxidase [45].Our results (Table 2 and Table 3) are not in agreements with other authors [18,27,45].Antibodies anti-hRAGE down-regulated ROS production in PBMNC either from T2DM or from healthy controls (Table 2, Table 3, Figure 1 and Figure 2).It can be due to experimental design we have used.Polyclonal antibodies anti-hRAGE could interact simultaneously with several epitopes on RAGE molecule while monoclonal antibodies or natural AGE interacts with a specific region or epitope.Several metabolic signaling simultaneously activated resulted in different metabolic response.In order to confirm our present results, we performed experiments using an NADPH inhibitor (DPI).The addition of NADPH oxidase inhibitor to PBMNC challenged with antibodies anti-hRAGE showed an addictive effect in reducing ROS production in PBMNC (Table 2 and Figure 1).Both PBMNC from T2DM patients and from healthy controls showed similar and comparable metabolic response in the presence of Ab anti-hRAGE.However the inhibition in hyperglycemia primed PBMN showed greater when compared to healthy control (p < 0.05) It suggests that the effect of Ab anti-hRAGE on RAGE can affected by hyperglycemia in diabetes.We studied down regulation of ROS production mediated by anti-hRAGE on PBMNC previously stimulated with a protein Kinase C (PKC) activator.Our results demonstrated that the activation of ROS production induced by PDB ( phorbol ester) was greater in PBMNMC from patients and was fully reversed by Ab anti-hRAGE in cells from T2DM and healthy controls in similar percentage of inhibition (55 and 53%, respectively) (Table 3 and Figure 2).It may suggests that the effect of antibodies anti-hRAGE (AGE-like) depends on PKC and /or NADPH-oxidase signaling pathways.Our present data demonstrated that polyclonal antibody anti-hRAGE induced ROS (oxidizing response) inhibition and increased cytokine secretion by PBMNC from T2DM.It suggests a dual effect for antibodies anti-hRAGE (AGE-like).Inflammatory changes observed in the presence of hyperglycemia have been associated with NF-kappaB activation [46,47].It is also described that RAGE activation leads to sustained and chronic activation of    NF-kappaB [32][33][34].Surprisingly, our results showed that antibodies anti-hRAGE (AGE-like) increased secretion of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in cultured PBMNCs from T2DM, but inhibited ROS generation.Secretion of TNF-alpha was stimulated in cells from both studied groups (Figure 3).The increase of IL-1β may suggest the involvement of inflammasome in diabetic complications.It has been reported that increased levels of TNF, IL-1β and expression of NRLP3-inflammasome are associated with endothelial dysfunction and progression of atherosclerosis [48][49][50].Ruscitti et al. [50] demonstrated that patients with diabetes and Rheumatoid Arthritis (T2D/RA) increase IL-β when compared with other groups.Antibodies anti-RAGE has no effect on periodontitis, but down-modulated renal complication in diabetic rats and improves neovascularization in the ischemic leg treatment in diabetic mice [50][51][52].The use of antibodies anti-RAGE leads to a controversial results and needs further studies.
Our results suggests that antibodies anti-hRAGE act as AGE-like ligand potentiating the inflammatory response activating different signaling pathways associated to cytokine secretion in cells from patients in comparison to that from healthy control.
It suggests a very complicated phenomenon with mechanism still not fully known.Thus, the use of experimental and therapeutic antibodies, such as, mono or polyclonal antibodies anti-RAGE, need to be used carefully and reinforce the suggestion that the role of AGE-RAGE in pathogenesis of diabetic complications is more complex than it seems.
In conclusion, the effect of antibody anti-hRAGE (AGE-like) in primed cells by hyperglycemia in diabetes may activates different signaling network when interact with RAGE on cells surface leading to a dual results associated with oxidizing response and pro-inflammatory cytokine secretion.It may have consequences on innate immunity.

Figure 1 :
Figure 1: Effects of Rabbit antibody anti human RAGE (Ab anti-hRAGE) and NADPH oxidase inhibitor [diphenyliodonium chloride (DPI)] on ROS production by human peripheral blood mononuclear cells (PBMNCs) from Type 2 diabetes patients (T2DM) and healthy controls.Each point represents the average of 10 experiments ± SD.RLU = Relative Light Units.

Figure 2 :
Figure 2: Effects of Rabbit antibody anti-human RAGE (Ab anti-hRAGE) on ROS production in PDB-stimulated human peripheral blood mononuclear cells from T2DM and healthy controls.Each point represents the average of 3 ± SD.RLU = Relative Light Units, PDB = phorbol 12,13-dibutyrate ester.

Table 1 :
Clinical and biochemical characteristics of the studied population.

Table 2 :
Effect of human RAGE antibody and NADPH oxidase inhibitor diphenyliodonium chloride on reactive oxygen species (ROS) production in human peripheral blood mononuclear cells from T2DM and healthy controls.n= 10 for each group; PBMNCs = peripheral blood mononuclear cells; Ab anti-hRAGE = polyclonal antibodies against human RAGE; DPI = diphenyliodonium chloride (NADPH oxidase inhibitor); RLU = Relative Light Units.* p < 0.05 vs. healthy controls (Student's t-test) Percentage inhibition values were calculated from the expression [1 − R2/R1)] × 100: a R1 and R2 represent ROS levels in the absence or presence of hRAGEab, respectively b R1 and R2 represent ROS levels in the presence of DPI and hRAGEab, respectively.P > 0.05 when similar letters were compared.