Francisco Blanco-Favela

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.

Analysis of anti-prolactin autoantibodies in systemic lupus erythematosus.

Evidence has shown that prolactin is an essential component of an effective immune response. In systemic lupus erythematosus, clinical trials have produced controversial information about the role of PRL. Some results find association between serum PRL levels and disease activity. In contrast, other authors did not find this. Recently, autoantibodies against prolactin in SLE patients have been described. One hundred percent of SLE patients with anti-PRL autoantibodies had hyperprolactinemia (hPRL) and 31.7% of the SLE patients classified with idiopathic hPRL had anti-prolactin antibodies. A similar result was found in 103 pediatric SLE patients. The patients with idiopathic hyperprolactinemia and anti-PRL autoantibodies had less clinical and serological lupus activity than the SLE patients with idiopathic hyperprolactinemia, but without anti-PRL autoantibodies. This evidence suggests that anti-PRL autoantibodies or the complex with any other molecule, like macroprolactinemia (big-big PRL) could have attenuated biological activity and this could explain why some clinical studies did not find any association between serum PRL levels and disease activity in SLE patients. However, studies in vitro have shown normal or elevated biological activity in Nb2 cell lines using PRL from serum with anti-PRL autoantibodies from patients with or without autoimmune diseases. Several conclusions could be drawn. One is that while a set of hyperprolactinemic SLE patients display autoantibodies against PRL, it is not clear what role these autoantibodies play in the whole system. However, until now, we knew that the patients with antibodies to PRL lacked the clinical symptoms of hyperprolactinemia such as menstrual disturbances and/or galactorrhea and show less clinical and serological lupus activity.

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.

Intradermal DNA vaccination in ear pinnae is an efficient route to protect cats against rabies virus

A DNA vaccine against rabies (pGQH) was administrated to cats in order to examine different administration routes. Four groups of three cats each were inoculated with pGQH as follows: group A, intramuscularly (IM), 100 microg; group B, intranasally (IN), 100 microg; group C, intradermally into ear pinnae (ID-EP), 100 microg, and group D, IM, 200 microL of phosphate buffer solution (PBS) alone (control group). Blood was drawn on days 0, 30, 60, 90, 120, 150, and 180. Groups A, B, and C received a booster on day 30. At day 200 all animals were challenged. A passive transfer of cat sera, as well as a viral challenge, was performed in mice. The results displayed that neutralizing antibody titers were higher in cats of group C (ID-EP) showing high early titers (> 2 IU) and the highest titer was on day 120 (> 14 IU). In group B (IN), two out of three cats seroconverted on day 30 (> 0.5 IU), the third cat seroconverted until day 60 (> 0.5 IU). In contrast, the lowest levels of neutralizing antibodies were detected in group A (IM). The control group showed no anti-rabies antibodies. Groups A (IM) and D (control) succumbed after lethal challenge. All animals from the ID-EP group (C) survived, only one individual from the IN (B) group died. Mice that received cat sera from ID-EP, IM, and IN groups survived and were protected (30/30 survivors). Mice groups that received pre-immunization sera from cats were not protected (0/30 survivors). This study demonstrates that pGQH immunization was successful when it was administrated ID-EP, and acceptable through the IN route. The IM route, however, was not effective in cats. For vaccination, the IN route seems attractive due to its accessibility for application, but it seems to activate seroconversion slowly. The best route to promote anti-rabies antibody titers was the ID-EP route. This practical and efficient route should be further studied.

Prolactin Levels Correlate with Abnormal B Cell Maturation in MRL and MRL/lpr Mouse Models of Systemic Lupus Erythematosus-Like Disease

Prolactin (PRL) plays an important role in modulating the immune response. In B cells, PRL enhances antibody production, including antibodies with self-specificity. In this study, our aims were to determine the level of PRL receptor expression during bone-marrow B-cell development and to assess whether the presence of high PRL serum concentrations influences absolute numbers of developing populations and disease outcome in lupus-prone murine models. We observed that the PRL-receptor is expressed in early bone-marrow B-cell; the expression in lupus-prone mice, which had the highest level of expression in pro-B cells and immature cells, differed from that in wild-type mice. These expression levels did not significantly change in response to hyperprolactinemia; however, populations of pro-B and immature cells from lupus-prone strains showed a decrease in the absolute numbers of cells with high PRL-receptor expression in response to PRL. Because immature self-reactive B cells are constantly being eliminated, we assessed the expression of survival factor BIRC5, which is more highly expressed in both pro-B and immature B-cells in response to PRL and correlates with the onset of disease. These results identify an important role of PRL in the early stages of the B-cell maturation process: PRL may promote the survival of self-reactive clones.

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.