Pro-inflammatory cytokines in patients with essential hypertension

BACKGROUND: It has been suggested that abnormalities in the immune response play a role in the pathogenesis of essential hypertension (EH). The aim of this study was to assess circulating concentrations and ex vivo production of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), and of the anti-inflammatory cytokines IL-1 receptor antagonist (IL-1ra) and IL-6, in patients with EH and compare them with healthy volunteers. PATIENTS AND METHODS: Plasma cytokine concentrations were measured in EH patients and control volunteers by specific radioimmunoassays and ELISA. Ex vivo cytokine production was assessed after stimulation of whole-blood with lipopolysaccharide. RESULTS: Circulating concentrations of TNF, IL-1 and IL-6 did not differ between EH patients and controls. In contrast, IL-1ra circulating levels were higher in EH patients. Hypertensive patients had an increased IL-1 and IL-6 production capacity when whole blood was stimulated ex vivo with lipopolysaccharide, while TNF production was lower. IL-1ra production capacity did not differ between patients and controls. CONCLUSIONS: Patients with EH have an altered profile of pro-and anti-inflammatory cytokines, consistent with monocyte activation in the circulation. The importance of these changes for the pathogenesis of EH and/or its secondary complications remains to be elucidated.     

The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans

In the mucosa, the immune pathways discriminating between colonizing and invasive Candida, thus inducing tolerance or inflammation, are poorly understood. Th17 responses induced by Candida albicans hyphae are central for the activation of mucosal antifungal immunity. An essential step for the discrimination between yeasts and hyphae and induction of Th17 responses is the activation of the inflammasome by C. albicans hyphae and the subsequent release of active IL-1β in macrophages. Inflammasome activation in macrophages results from differences in cell-wall architecture between yeasts and hyphae and is partly mediated by the dectin-1/Syk pathway. These results define the dectin-1/inflammasome pathway as the mechanism that enables the host immune system to mount a protective Th17 response and distinguish between colonization and tissue invasion by C. albicans.     

Receptor Recognition of and Immune Intracellular Pathways for Veillonella parvula Lipopolysaccharide

Veillonella parvula is an anaerobic gram-negative coccus that is part of the normal flora of the animal and human mouth and gastrointestinal and genitourinary tracts. Oral V. parvula is involved in the development of early periodontal disease as well as different types of serious infections. Present data on molecular mechanisms responsible for innate immune response against Veillonella are very scanty. The aim of this study was to investigate the Toll-like receptor (TLR) pathways responsible for V. parvula lipopolysaccharide (LPS) and to identify the intracellular pathways induced by this recognition. V. parvula LPS stimulated tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) release in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Pretreatment of cells with a TLR4 antagonist significantly reduced TNF-α and IL-6 production in PBMC stimulated with either Veillonella or Escherichia coli LPS. However, V. parvula LPS was 10- to 100-fold less active than E. coli LPS for cytokine induction. TNF-α, IL-1β, IL-6, and IL-10 were released in wild-type and TLR2^−/−, but not TLR4^−/−, mouse macrophage cultures. V. parvula LPS was able to activate the human PBMC p38 mitogen-activated protein kinase (MAPK). A specific p38 MAPK inhibitor strongly inhibited V. parvula LPS-induced TNF-α, IL-1β, IL-6, and IL-10. In conclusion, V. parvula LPS is able to induce cytokine production in both human and murine in vitro models, although it is less effective than Enterobacteriaceae LPS. V. parvula LPS-stimulated cytokine induction, as well as p38 MAPK activation, are TLR4-dependent features.Veillonella organisms are small, nonfermentative, strictly anaerobic, gram-negative cocci which form part of the normal flora of the oral, genitourinary, respiratory, and intestinal tracts of humans and animals (10). The genus Veillonella was first isolated by Veillon and Zuber in 1898 and currently consists of eight species (28).Veillonella species have been reported as causes of serious infections, including meningitis (6), osteomyelitis and discitis (7, 28), prosthetic joint infection (26), and acute and chronic pleuropulmonary infection (33).Risk factors for Veillonella infection include periodontal disease, immunodeficiency, intravenous drug use, and premature birth (28). V. parvula is an important pathogen implicated in periodontitis and other dental infections (3, 18), and it is one of the most common anaerobic pathogens in chronic maxillary sinusitis and deep neck infections (9, 37). V. parvula has also been reported as a pathogen for osteomyelitis (34) and abscessed orchiepididymitis with sepsis (4). Endovascular infections reportedly may range from bacteremia to severe endocarditis and fatal cases of sepsis (8, 14, 25).Lipopolysaccharides (LPS) are major pathogenic factors of gram-negative bacteria. LPS from aerobic and facultative bacteria have been extensively studied (5). On the contrary, very little is known regarding the biological activity of LPS from anaerobic microorganisms such as Veillonella (10, 24, 29, 32). In addition, little is known about cellular and molecular mechanisms responsible for innate immune response against V. parvula, as well as for inflammatory reactions leading to severe periodontitis or sinusitis. Toll-like receptors (TLRs) recognize microbial compounds (36) and trigger the inflammatory and immune responses against pathogens. Immunohistochemical localization of TLR2 and TLR4 in gingival tissue of periodontitis patients has been reported (30). In mammals, engagement of TLRs by LPS results in the recruitment of cytoplasmic signaling molecules (12, 36), which eventually activates mitogen-activated protein kinases (MAPKs), including p38, JNK, and extracellular signal-regulated kinase (ERK). MAPK activation leads to cytokine release (2).However, the interaction between oral V. parvula LPS and TLRs has not been directly studied yet. The aim of this study was to investigate the potential role of TLR2 and TLR4 for the recognition of Veillonella parvula LPS in both human peripheral blood mononuclear cells (PBMC) and in TLR2 and TLR4 knockout (KO) mouse macrophages, as well as the intracellular kinase signaling pathways induced after challenge of monocytes with Veillonella parvula LPS.     

Modulation of muramyl dipeptide stimulation of cytokine production by blood components

Muramyl dipeptide (MDP) is the minimal active fragment of peptidoglycan of the cell wall of Gram‐positive bacteria, with potential beneficial effects as a vaccine adjuvant. Peptidoglycans and MDP are recognized by the intracellular receptor NOD2 (nucleotide‐binding oligomerization domain 2), leading to production of proinflammatory cytokines. In the present study, it is shown that, despite stimulatory effects on isolated human mononuclear cells, MDP does not stimulate production of tumour necrosis factor‐α, interleukin‐1β or interleukin‐6 in a whole‐blood assay. However, MDP retains synergistic effects on lipopolysaccharide‐induced cytokines in whole blood. Screening tests of NOD2 function based on whole‐blood stimulation should therefore employ strategies based on the synergistic effects of MDP on Toll‐like receptor‐induced cytokine production. Plasma was not responsible for the inhibition of MDP in whole blood. The inhibition of MDP stimulation was dependent upon cellular components, with erythrocyte‐derived haemoglobin and neutrophils collaborating in the inhibition of MDP effects in whole blood.     

Disease-specific ex vivo stimulation of whole blood for cytokine production: applications in the study of tuberculosis

As a simple method for field studies to assess the cytokine-status of patients with tuberculosis (TB), the use of whole blood instead of isolated cells has advantages, especially since the risk of contamination is minimal. Therefore, cytokine production in whole blood cultures was determined using non-specific and disease-specific stimuli. Heparinized blood from healthy volunteers was either incubated in closed vacutainer tubes or in tissue culture wells after dilution in culture medium. Dose-response and kinetics were investigated for the production of TNFalpha, IL-1beta, IL-1ra, IL-10 and IFNgamma. Patients with TB and healthy individuals were examined for IFN-gamma production in whole blood. In the absence of a stimulus, the production of cytokines is negligible in whole blood cultures. LPS induces the production of TNFalpha, IL-1beta, IL-1ra and IL-10; PHA induces the production of IFNgamma and IL-10. Live BCG induces the production of proinflammatory cytokines, irrespective of tuberculin skin status. In contrast, PPD and MTB-culture filtrate induce production of IFNgamma in skin-test positive and not in skin-test negative healthy subjects. Five out of 13 patients with TB had a low antigen-specific IFNgamma production, suggestive of a minimal or absent specific T-cell response. For most purposes, cultures in closed vacutainer tubes are optimal. If one wishes to focus on T-cell cytokines or if only small volumes of blood are available, dilution of whole blood in culture medium before incubation in tissue culture wells may be preferable.     

The impact of caspase-12 on susceptibility to candidemia

Candida is one of the leading causes of sepsis, and an effective host immune response to Candida critically depends on the cytokines IL-1β and IL-18, which need caspase-1 cleavage to become bioactive. Caspase-12 has been suggested to inhibit caspase-1 activation and has been implicated as a susceptibility factor for bacterial sepsis. In populations of African descent, CASPASE-12 is either functional or non-functional. Here, we have assessed the frequencies of both CASPASE-12 alleles in an African-American Candida sepsis patients cohort compared to uninfected patients with similar predisposing factors. African-American Candida sepsis patients (n = 93) and non-infected African-American patients (n = 88) were genotyped for the CASPASE-12 genotype. Serum cytokine concentrations of IL-6, IL-8, and IFNγ were measured in the serum of infected patients. Statistical comparisons were performed in order to assess the effect of the CASPASE-12 genotype on susceptibility to candidemia and on serum cytokine concentrations. Our findings demonstrate that CASPASE-12 does not influence the susceptibility to Candida sepsis, nor has any effect on the serum cytokine concentrations in Candida sepsis patients during the course of infection. Although the functional CASPASE-12 allele has been suggested to increase susceptibility to bacterial sepsis, this could not be confirmed in our larger cohort of fungal sepsis patients.     

Mechanical ventilation in healthy mice induces reversible pulmonary and systemic cytokine elevation with preserved alveolar integrity: an in vivo model using clinical relevant ventilation settings

BACKGROUND: Mechanical ventilation (MV) may activate the innate immune system, causing the release of cytokines. The resulting proinflammatory state is a risk factor for ventilator-induced lung injury. Cytokine increase results from direct cellular injury but may also result from cyclic stretch alone as demonstrated in vitro: mechanotransduction. To study mechanotransduction in vivo, the authors used an animal MV model with clinically relevant ventilator settings, avoiding alveolar damage. METHODS: Healthy C57BL6 mice (n = 82) were ventilated (tidal volume, 8 ml/kg; positive end-expiratory pressure, 4 cm H2O; fraction of inspired oxygen, 0.4) for 30, 60, 120, and 240 min. Assigned animals were allowed to recover for 2 days after MV. Both pulmonary tissue and plasma interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor alpha, IL-6, IL-10, and keratinocyte-derived chemokine levels were measured. Histopathologic appearance of lung tissue was analyzed by light microscopy and electron microscopy. RESULTS: In lung tissue, all measured cytokines and keratinocyte-derived chemokine levels increased progressively with MV duration. Light microscopy showed increased leukocyte influx but no signs of alveolar leakage or albumin deposition. Electron microscopy revealed intact epithelial cell and basement membranes with sporadically minimal signs of partial endothelial detachment. In plasma, increased levels of IL-1alpha, tumor necrosis factor alpha, IL-6, and keratinocyte-derived chemokine were measured after MV. In the recovery animals, cytokine levels had normalized and no histologic alterations could be found. CONCLUSIONS: Mechanical ventilation induces reversible cytokine increase and leukocyte influx with preserved tissue integrity. This model offers opportunities to study the pathophysiologic mechanisms behind ventilator-induced lung injury and the contribution of MV to the "multiple-hit" concept.