Karina Chávez-Rueda

The activation of CD14, TLR4, and TLR2 by mmLDL induces IL-1β, IL-6, and IL-10 secretion in human monocytes and macrophages

Atherosclerosis is considered a chronic inflammatory disease in which monocytes and macrophages are critical. These cells express CD14, toll-like receptor (TLR) 2, and TLR4 on their surfaces, are activated by minimally modified low-density lipoprotein (mmLDL) and are capable of secreting pro-inflammatory cytokines. The aim of this research was thus to demonstrate that the activation of CD14, TLR2, and TLR4 by mmLDL induces the secretion of cytokines.MethodsHuman monocytes and macrophages were incubated with monoclonal antibodies specific for CD14, TLR4, and TLR2 prior to stimulation with mmLDL. Cytokine secretion was then compared to that observed upon mmLDL stimulation in untreated cells.ResultsStimulation with mmLDL induced the secretion of pro-inflammatory cytokines. Blocking CD14 in monocytes inhibited secretion of interleukin (IL)-1β (72%), IL-6 (58%) and IL-10 (63%), and blocking TLR4 inhibited secretion of IL-1β by 67%, IL-6 by 63% and IL-10 by 60%. Blocking both receptors inhibited secretion of IL-1β by 73%, IL-6 by 69% and IL-10 by 63%. Furthermore, blocking TLR2 inhibited secretion of IL-1β by 65%, IL-6 by 62% and IL-10 by 75%. In macrophages, we found similar results: blocking CD14 inhibited secretion of IL-1β by 59%, IL-6 by 52% and IL-10 by 65%; blocking TLR4 inhibited secretion of IL-1β by 53%, IL-6 by 63% and IL-10 by 61%; and blocking both receptors inhibited secretion of IL-1β by 69%, IL-6 by 67% and IL-10 by 65%. Blocking TLR2 in macrophages inhibited secretion of IL-1β by 57%, IL-6 by 40% and IL-10 by 72%.ConclusionOur study demonstrates that CD14, TLR4, and TLR2 participate in the immune response against mmLDL by inducing the production of pro-inflammatory cytokines in both monocytes and macrophages. These findings suggest that the activation of these receptors by mmLDL contributes to the inflammatory process of atherosclerosis.

Innate Immune System Cells in Atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by innate and adaptive immune system involvement. A key component of atherosclerotic plaque inflammation is the persistence of different innate immune cell types including mast cells, neutrophils, natural killer cells, monocytes, macrophages and dendritic cells. Several endogenous signals such as oxidized low-density lipoproteins, and exogenous signals such as lipopolysaccharides, trigger the activation of these cells. In particular, these signals orchestrate the early and late inflammatory responses through the secretion of pro-inflammatory cytokines and contribute to plaque evolution through the formation of foam cells, among other events. In this review we discuss how innate immune system cells affect atherosclerosis pathogenesis.

Activation of TLR2 and TLR4 by minimally modified low-density lipoprotein in human macrophages and monocytes triggers the inflammatory response.

Oxidized low-density lipoproteins and Toll-like receptors (TLR) 2 and 4 are involved in the development of atherosclerosis. The TLR are important in the pro-inflammatory response. The aim of this research was to analyze the activation of CD14, TLR4, and TLR2 in response to minimally modified low-density lipoprotein (mmLDL). Human monocytes and macrophages secreted tumor necrosis factor (TNF)-alpha in response to mmLDL, and blocking CD14 or TLR4 resulted in a approximately 60% decrease in mmLDL-induced TNF-alpha secretion. We also observed similar inhibition of TNF-alpha synthesis in human monocytes ( approximately 65%) and macrophages ( approximately 70%) when both receptors were blocked simultaneously. When TLR2 was blocked, TNF-alpha synthesis was inhibited by approximately 70% in both cell types. Moreover mmLDL induced redistribution of CD14, TLR4, and TLR2 on the cell surface. This is the first evidence that TLR2 and TLR4 are upregulated in response to mmLDL. Our results suggest that mmLDL activates CD14, TLR4, and TLR2, inducing the production of TNF-alpha and increasing the expression of TLR2 and TLR4.

The role of TLR2, TLR4 and CD36 in macrophage activation and foam cell formation in response to oxLDL in humans.

UNLABELLED Toll-like receptor (TLR)2, TLR4 and CD36 are central in inflammation and the development of atherosclerosis. Oxidized low-density lipoprotein (oxLDL) plays a critical role in this disease through its involvement in the formation of foam cells and the activation of leukocytes. The aim of this research was to analyze the role of TLR2, TLR4 and CD36 in foam cell differentiation and macrophage activation. METHODS Human macrophages were incubated with monoclonal antibodies specific for TLR2, TLR4 and CD36 prior to stimulation with oxLDL. Subsequently, we analyzed foam cell formation, cytokine secretion, histocompatibility complex (MHC) class II molecules and CD86 expression and T cell proliferation. RESULTS The stimulation of macrophages with oxLDL induced foam cell formation, cytokine secretion, HLA-DR and CD86 expression and T cell proliferation. The blockage of TLR2, TLR4 and CD36 reduced the secretion of IL-1β, IL-6 and IL-8, the expression of HLA-DR and CD86, T cell proliferation and foam cell formation. However, the blockage of TLR2 did not affect the formation of foam cells. CONCLUSION Our study demonstrates that TLR2, TLR4 and CD36 participate in the immune response to oxLDL by inducing an increase in pro-inflammatory cytokines, the expression HLA-DR and CD86 and the proliferation of T cells. However, TLR2 does not participate in the formation of foam cells, while TLR4 and CD36 play a relevant role in this process. These findings suggest that the activation of these receptors by oxLDL contributes to the pathogenesis of atherosclerosis.

Biologic activity and plasma clearance of prolactin–IgG complex in patients with systemic lupus erythematosus

OBJECTIVE To characterize the clinical findings in hyperprolactinemic systemic lupus erythematosus (SLE) patients with or without macroprolactinemia (big, big prolactin [PRL]) due to anti-PRL autoantibodies (PRL-IgG complex), and to assess the bioactivity and structure of big, big PRL. METHODS Twenty-seven SLE patients with hyperprolactinemia (HPRL) were studied. Patients with (n = 8) or without (n = 19) big, big PRL were identified by gel filtration chromatography and affinity chromatography for IgG. PRL concentrations in serum and fractions by gel filtration chromatography and affinity chromatography were characterized by immunoradiometric assay (IRMA), Nb2 bioassay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, and clearance studies. RESULTS SLE patients without big, big PRL had significantly higher levels of disease activity, cause-proven HPRL, and menstrual disturbances compared with patients with big, big PRL (P < or = 0.05). The big, big PRL fractions by Western blotting revealed a single 23-kd nonglycosylated PRL. The Nb2:IRMA ratio of the samples with big, big PRL was significantly higher than that of the samples without big, big PRL (P < or = 0.02). However, bioactivity of big, big PRL in the Nb2 cells was very similar to that of 23-kd nonglycosylated PRL. Clearance studies in rats demonstrated that the PRL-IgG complex was eliminated more slowly than monomeric PRL (little PRL). CONCLUSION We demonstrated that the PRL-IgG complex was formed by 23-kd nonglycosylated PRL that was noncovalently bound to IgG and showed that the complex was fully active in vitro. This result suggests that the absence of symptoms of HPRL or lower levels of lupus activity in these patients is not explained by lower bioactivity of the complex. Instead, because of the large molecular size of the complex, the PRL does not easily cross the capillary walls. Delayed clearance of the PRL-IgG complex may account for increased serum levels of PRL in SLE patients with anti-PRL autoantibodies.

Effect of prolactin on lymphocyte activation from systemic lupus erythematosus patients.

Abstract:  The aim was to explore the role of prolactin (PRL) in the lymphocyte activation process in systemic lupus erythematosus (SLE) patients in an in vitro model. Peripheral blood mononuclear cells (PBMCs) were isolated from SLE patients and healthy individuals. The mRNA for PRL and its receptor obtained by standard techniques, with an appropriate primer, were subjected to polymerase chain reaction (PCR) and visualized. The PBMCs were cultured with (a) medium alone as a negative control, (b) unspecific mitogen as a positive control, (c) PRL alone, (d) mitogen plus PRL, (e) mitogen plus antibody anti‐PRL, and (f) mitogen plus a nonrelated antibody. Then CD69 and CD154 were determined by flow cytometry analysis. Twelve inactive and 15 active SLE patients were studied. Twenty‐five percent of the active patients displayed hyperprolactinemia. Under basal conditions CD69 expression was associated with disease activity. The PBMCs activated in vitro were capable of producing and secreting PRL, measured by mRNA and Nb2 assay. In a similar way, the mRNA for the PRL receptor was visualized. Cells from SLE patients cultivated with PRL alone did not display increased CD69 and CD154 expression. The addition of PRL to the unspecific stimulated culture does not have an additive effect. In contrast, the addition of antibodies against PRL in order to block the autocrine PRL resulted in a striking reduction of CD69 and CD154 expression.

Prolactin down-regulates CD4+CD25hiCD127low/− regulatory T cell function in humans

Among its many functions, prolactin (PRL) participates in immune responses and promotes the activation, differentiation and proliferation of T cells. However, the mechanisms by which PRL regulates regulatory T (T(reg)) cells are still unknown. Our goal was to determine whether PRL plays a role in T(reg) function. We measured the expression of PRL and its receptor in T(reg) and effector T (T(eff)) cells from 15 healthy individuals. We also evaluated the functional activity of T(reg) cells by examining proliferation and cytokine secretion in cells activated with anti-CD3/CD28 in the presence or absence of PRL. We report that T(reg) cells constitutively expressed PRL receptor, whereas T(eff) cells required stimulation with anti-CD3/CD28 to induce PRL receptor expression. Expression of PRL was constitutive in both populations. We found that the addition of PRL inhibited the suppressor effect (proliferation) mediated by T(reg) cells in vitro, reducing suppression from 37.4 to 13% when PRL was added to co-cultures of T(reg) and T(eff) cells (P<0.05). Cultures treated with PRL favoured a Th1 cytokine profile, with increased production of TNF and IFNγ. We report for the first time that PRL receptor expression was constitutive in T(reg) cells but not in T(eff) cells, which require stimulation to induce PRL receptor expression. PRL inhibited the suppressive function of T(reg) cells, apparently through the induced secretion of Th1 cytokines.

Function of Treg Cells Decreased in Patients With Systemic Lupus Erythematosus Due To the Effect of Prolactin

Abstract Prolactin has different functions, including cytokine secretion and inhibition of the suppressor effect of regulatory T (Treg) cells in healthy individuals. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by defects in the functions of B, T, and Treg cells. Prolactin plays an important role in the physiopathology of SLE. Our objective was to establish the participation of prolactin in the regulation of the immune response mediated by Treg cells from patients with SLE. CD4+CD25hiCD127−/low cells were purified using magnetic beads and the relative expression of prolactin receptor was measured. The functional activity was evaluated by proliferation assay and cytokine secretion in activated cells, in the presence and absence of prolactin. We found that both percentage and function of Treg cells decrease in SLE patients compared to healthy individuals with statistical significance. The prolactin receptor is constitutively expressed on Treg and effector T (Teff) cells in SLE patients, and this expression is higher than in healthy individuals. The expression of this receptor differs in inactive and active patients: in the former, the expression is higher in Treg cells than in Teff cells, similar to healthy individuals, whereas there is no difference in the expression between Treg and Teff cells from active patients. In Treg:Teff cell cocultures, addition of prolactin decreases the suppressor effect exerted by Treg cells and increases IFN&ggr; secretion. Our results suggest that prolactin plays an important role in the activation of the disease in inactive patients by decreasing the suppressor function exerted by Treg cells over Teff cells, thereby favoring an inflammatory microenvironment.

Prolactin promoter polymorphism (-1149 G/T) is associated with anti-DNA antibodies in Mexican patients with systemic lupus erythematosus.

Prolactin (PRL) is a 23-kDa protein hormone that is synthesized mainly by the anterior pituitary gland. However, PRL can also be synthesized and secreted by extrapituitary tissues, particularly immune cells. A biallelic polymorphism (−1149 G/T) in the prolactin promoter has been shown to be functionally important, as modulation of prolactin expression has been associated with SLE in some populations. We have performed an association study using Mexican patients with SLE. We used qPCR to determine the SNP allele and genotype frequencies. We did not find statistically significant differences in allele and genotype frequencies between patients and healthy controls. However, we found a statistically significant association between the G allele and the presence of anti–dsDNA antibodies in serum (Allele frequency (G): P = 0.005; Genotyping frequency (GG): P = 0.001, OR = 7.8, 95% CI 3.59–27.1). Our data demonstrate that the prolactin promoter polymorphism −1149 G/T does not significantly contribute to SLE disease susceptibility but does predispose carriers to other immunological changes.

Intranasal Anti-rabies DNA Immunization Promotes a Th1-related Cytokine Stimulation Associated with Plasmid Survival Time

BACKGROUND AND AIMS DNA vaccination has a great potential to decrease infectious diseases worldwide, such as rabies. Here we showed the effects of a single anti-rabies DNA vaccination applied intranasally (IN) on plasmid survival time, neutralizing antibody (NA) titers, G-protein expression and Th1/Th2-related cytokines. METHODS Only one 50-μg dose of an anti-rabies DNA vaccine was IN administered to 160 Balb/c mice. Twenty mice were used for the neutralizing antibody study, 35 for the proliferation assay, 35 for Th1/Th2-related cytokines, 35 for glycoprotein expression by immunocytochemistry, and 35 for pGQH detection and G-protein mRNA expression. RESULTS Th1-type related cytokines from spleen cells (IFN-γ, TNF-α, and IL-2) were detected. Rabies NA titers were ≥0.6 IUs from day 30 onward in the IN DNA-vaccinated group. The plasmid was identified in brains and lungs from days 3-15. The mRNA transcript was amplified in brains and lungs from days 3-30, and G-protein expression was observed in spleens, brains and lungs on days 3, 8, and 15. In all cases, a gradual decrease was observed on days 30 and 45 and absent on day 60. CONCLUSIONS We found that Th1-type related cytokines (IL-2, IFN-γ, and TNF-α) were stimulated during the first month after DNA vaccination, correlating with the proliferation assays. Also, it was associated with the plasmid survival time remaining in lungs and brains prior to its degradation.