Anna Pietrzak

Surface TLR2 and TLR4 Expression on Mature Rat Mast Cells Can Be Affected by Some Bacterial Components and Proinflammatory Cytokines

The aim of our study was to determine whether some bacterial components as well as some proinflammatory cytokines can affect surface mast cell levels. By the use of flow cytometry technique, we documented that freshly isolated mature rat peritoneal mast cells do express surface TLR2 and TLR4 protein, but not CD14 molecules, and respond to stimulation with TLR2 and TLR4 ligands by cysteinyl leukotriene generation. The level of TLR2 protein is modulated by PGN and CCL5 treatment, but not by LPS, LAM, TNF, or IL-6. Surface mast cell TLR4 expression is affected by LPS, LAM, IL-6, and CCL5. Considering that TLR-mediated activation conditions not only engaged these cells in antibacterial defense and development of inflammation but also might influence allergic processes, our observations that surface TLR2 and TLR4 expression can be regulated both bacterial components and proinflammatory cytokines seem to be very intriguing and importance.

Interleukin (IL)-10 inhibits RANTES-, tumour necrosis factor (TNF)- and nerve growth factor (NGF)-induced mast cell migratory response but is not a mast cell chemoattractant

Interleukin (IL)-10 is an important immunoregulatory cytokine with multiple biologic effects on different cell types. This cytokine also affects mast cell development, survival and activity. Mast cells are well known for their role in diverse pathophysiological processes including inflammatory events. Mast cell number in tissues is high and relatively constant. However, it is well established that these cells accumulate at the sites of inflammation in response to chemoattractants, e.g. RANTES, tumour necrosis factor (TNF) and nerve growth factor (NGF). In the present study, we examined whether IL-10 influenced RANTES-, TNF- and NGF-induced rat peritoneal mast cell migration. We also studied whether IL-10 could act as mast cell chemoattractant. We provided evidence, for the first time ever, that IL-10 influenced mature mast cell migration, i.e. it strongly decreased RANTES-induced mast cell migration and completely inhibited mast cell migratory response to TNF and NGF. The effective concentration of IL-10 that inhibited RANTES-, TNF- and NGF-induced mast cell migratory response was in the nanomolar range. The inhibitory effect of IL-10 on cytokine-stimulated mast cell migration was specific, as it was completely blocked by anti-IL-10R antibodies, and STAT3-dependent. In addition, our results have shown that IL-10 was not a mast cell chemoattractant. Thus, our findings clearly demonstrated that IL-10 may affect mast cell number within tissue by inhibiting local mast cell accumulation stimulated by chemotactic factors.

Tumor necrosis factor α (TNF-α) activates human adenoidal and cutaneous mast cells to histamine secretion

Abstract It is widely known that mast cells and cytokine TNF-α are both involved in inflammatory reactions. Therefore, we have studied whether TNF-α can cause histamine secretion from human adenoidal and cutaneous mast cells. The experiments were performed in vitro on mast cells isolated from tissues by enzymatic dispersion technique. The results of our experiments have clearly shown that this cytokine stimulates mast cells to histamine release. TNF-α-induced histamine release was concentration- and time-dependent. Moreover, the release of histamine evoked by TNF-α was also dependent on reaction temperature and on glycolytic and oxidative cellular metabolism. We have concluded that TNF-α is a potent stimulus for mast cells to release histamine and that it induces histamine release via an active, secretory process.

Action of Tumor Necrosis Factor-alpha on Rat Mast Cells

Taking into account that cytokine tumor necrosis factor-alpha (TNF-alpha) and mast cells (MC) both are involved in inflammation, it seems of great importance to recognize their relationships. Therefore, we have studied whether recombinant human TNF-alpha (rHuTNF-alpha) can cause histamine secretion from rat peritoneal MC. We have also examined the effect of this cytokine on MC reactivity. We have established that TNF-alpha stimulates rat MC to histamine release in a concentration-dependent manner. TNF-alpha-induced histamine secretion was evoked by concentrations > 10-16 M and reached the maximum rate at a concentration of 10-10 M (histamine release 17.1% +/- 1.9%, mean +/- SEM). We have also noticed that pretreatment of MC with TNF-alpha (in a concentration of 10-16 M) significantly inhibited concanavalin A (ConA)-stimulated release of histamine, with the percent release decreasing to 51% of the control value. Treatment of mast cells with TNF-alpha resulted in a decrease of compound 48/80-dependent histamine release as well (the percent released histamine fell to 85% of the control value). This altered MC responsiveness was reversible. After 120 min of resting time, the MC reactivity came back to the initial values. We have concluded that TNF-alpha appears to be a direct stimulus for MC to release histamine, and it may regulate MC secretory function.

Cathelicidin rCRAMP stimulates rat mast cells to generate cysteinyl leukotrienes, synthesize TNF and migrate: involvement of PLC/A2, PI3K and MAPK signaling pathways

Cathelicidins represent a family of cationic peptides involved in host defense systems. Apart from exerting direct anti-microbial effects, cathelicidins can regulate immune responses by affecting the activity of cells playing a role in antibacterial defense. Taking into account that mast cells are critical components of host defense, the aim of this study was to determine whether rat cathelicidin-related anti-microbial peptide (rCRAMP) can influence mast cell activity. We have demonstrated that activation of fully mature rat mast cells with rCRAMP resulted in generation and release of cysteinyl leukotrienes (cysLTs). However, rCRAMP failed to induce mast cell degranulation and histamine release. We also found that rCRAMP stimulated rat mast cells to synthesize TNF, but not CXCL8. What is more, this peptide induced GM-CSF, IL-1β, CCL2 and CCL3 but not IL-33 mRNA expression in mast cells. Finally, we showed that this cathelicidin serves as potent chemoattractant for rat mast cells. rCRAMP-mediated cysLT synthesis and mast cell migration were strongly inhibited by IL-10 pre-treatment. With the use of specific inhibitors, we established that activation of PLC/A2 and ERK1/2, but not p38, was required for rCRAMP-induced mast cell stimulation, while PI3K-dependent pathway is involved in both TNF synthesis and mast cell migration. Our results suggest that cathelicidins can amplify inflammatory responses by causing mast cells accumulation and by stimulating these cells to release potent pro-inflammatory mediators.

Different potency of bacterial antigens TLR2 and TLR4 ligands in stimulating mature mast cells to cysteinyl leukotriene synthesis.

The aim of study was to compare the potency of different bacterial antigens to induce rat mature mast cell to cysteinyl leukotriene (cysLT) generation. We examined Toll‐like receptor (TLR)2 agonists, i.e. lipoteichoic acid (LTA) Staphylococcus faecalis, Streptococcus pyogenes, Bacillus subtilis and Staphylococcus aureus, lipoarabinomannan (LAM) Mycobacterium smegmatis, peptydoglican (PGN) Staphylococcus aureus, as well as TLR4 agonists, i.e. lipopolysaccharide (LPS) Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Pophyromonas gingivalis and Escherichia coli. We also estimated the effect of tumor necrosis factor (TNF)‐, interleukin (IL)‐6‐, CCL5‐, and IL‐10‐priming on mast cell cysLT synthesis following bacterial antigen activation. We found that all bacterial antigens activated mast cells to cysLT generation; however, the extent of cysLT release in response to stimulation varied. Out of the examined antigens LPS P. gingivalis exhibited the highest potency, as it induced cysLT generation acting at a very low concentration (10−4 ng/mL). Other LPSs affected mast cells at higher (up to 105‐fold) concentrations. LTAs were the most effective at concentrations of 5 × 102 ng/mL, while LAM and PGN stimulated mast cells to maximal cysLT generation at concentrations as high as 105 ng/mL. Anti‐TLR2 and anti‐TLR4 antibodies, as well as nuclear factor κB (NF‐κB) inhibitor significantly diminished cysLT generation in response to bacterial antigen stimulation. Priming with TNF, IL‐6 and CCL5 did not affect bacterial antigen‐induced cysLT generation, while IL‐10‐pretreatment caused significant decrease in cysLT synthesis by mast cells. These observations might have a great pathophysiological importance; inasmuch cysLTs strongly influence the development and intensity of inflammation during bacterial infection.