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Background: The effect of a docosahexaenoic acid (DHA)-rich fish oil (FO) supplementation on human leukocyte function was investigated., Methods: Ten male volunteers were supplemented with 3g/day FO containing 26% eicosapentaenoic acid (EPA, 20:5, n-3) and 54% DHA (22:6, n-3) for 2 months., Results: FO supplementation changed the fatty acid (FA) composition of leukocytes resulting in an increase of n-3/n-6 ratio from 0.18 to 0.62 in lymphocytes and from 0.15 to 0.70 in neutrophils. DHA-rich FO stimulated an increase in phagocytic activity by 62% and 145% in neutrophils and monocytes, respectively. Neutrophil chemotactic response was increased by 128%. The rate of production of reactive oxygen species by neutrophils was also increased, as it was with lymphocyte proliferation. These changes were partially reversed after a 2-month wash out period. With respect to cytokine production by lymphocytes, interleukin (IL)-4 release was not altered, whereas secretions of IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha were raised. These results are in contrast to those described by others using EPA-rich FO supplementation. Lymphocyte pleiotropic gene expression was analyzed by a macroarray technique. Of the analyzed genes (588 in total), 77 were modified by the supplementation. FO supplementation resulted in up-regulation of 6 genes (GATA binding protein 2, IL-6 signal transducer, transforming growth factor alpha, TNF, heat shock 90kDa protein 1-alpha and heat shock protein 70kDa 1A) and a down regulation of 71 genes (92.2% of total genes changed). The largest functional group of altered genes was that related to signaling pathways (22% of the total modified genes)., Conclusions: Therefore, although EPA and DHA are members of n-3 FA family, changes in the proportion of DHA and EPA exert different effects on neutrophil, monocyte and lymphocyte function, which may be a result of specific changes in gene expression.

Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom. Previous work of our group demonstrated that this toxin or its phospholipase A(2) subunit inhibits macrophage spreading and phagocytosis. The phagocytic activity of macrophages is controlled by the rearrangement of actin cytoskeleton and activity of the small Rho GTPases. The effect of crotoxin and its subunit on actin reorganization and tyrosine phosphorylation in rat peritoneal macrophages, during phagocytosis of opsonized zymosan, was presently investigated. The crude venom was used as positive control. In addition, the effect of crotoxin on the activity of Rho and Rac1 small GTPases was examined. Transmission electron studies showed that the venom or crotoxin decreased the extent of spread cells and increased microprojections often extended from macrophage surface. Immunocytochemical assays demosntrated that the venom or toxins increased F-actin content in the cytoplasm of these cells, but induced a marked decrease of phosphotyrosine. These effects were abolished by treatment with zileuton, a 5-lipoxygenase inhibitor. Furthermore, crotoxin decreased membrane-associated RhoA and Rac1 in translocation assays. The present results indicate that the crotalid venom and crotoxin are able to induce cytoskeleton rearrangement in macrophages. This effect is associated with inhibition of tyrosine phosphorylation and of the activity of proteins involved in intracellular signalling pathways important for the complete phagocytic activity of these cells.

Crotalus durissus terrificus snake venom and its major toxin, crotoxin or type II PLA2 subunit of this toxin, induce an inhibitory effect on spreading and phagocytosis in 2h incubated macrophages. The involvement of arachidonate-derived mediators on the inhibitory action of the venom or toxins on rat peritoneal macrophage phagocytosis was presently investigated. Peritoneal cells harvested from naive rats and incubated with the venom or toxins or harvested from the peritoneal cavity of rats pre-treated with the toxins were used. Zileuton, a 5-lipoxygenase inhibitor but not indomethacin, a cyclooxygenase inhibitor, given in vivo and in vitro abolished the inhibitory effect of venom or toxins on phagocytosis. Resident peritoneal macrophages incubated with the venom or toxins showed increased levels of prostaglandin E2 and lipoxin A4, with no change in leukotriene B4. These results suggest that lipoxygenase-derived eicosanoids are involved in the inhibitory effect of C.d. terrificus venom, crotoxin or PLA2 on macrophage phagocytosis.

Objective and Design: In the present study, the effect of a synthetic peptide (H(92)-G(102)) identical to the C-terminus of murine S100A9 (mS100A9p) was investigated on adherent peritoneal cell function., Materials and Methods: For in vitro assays, peritoneal cells were obtained from the abdominal cavity of mice and incubated, with the different concentrations of mS100A9p, for 1 h, and then their spreading and phagocytosis activities were evaluated. For ex-vivo assays, cells obtained from animals treated for 1 h with the peptide were submitted to the mannose-receptor phagocytosis assay. Shorter homologue peptides to the C-terminus of mS100A9p were also evaluated on in vitro phagocytosis assays of Candida albicans particles., Results: mS100A9p reduced both the spreading index and phagocytic activity, in vitro and ex-vivo, independent of the receptor evaluated. The homologue peptide corresponding to the H(92)-E(97) region of mS100A9p, the zinc-binding motif, was responsible for such an effect., Conclusion: These results suggest a modulator effect of the C-terminus of S100A9 protein on the function of adherent peritoneal cells.

Recent work demonstrated that crotoxin, the main toxin of Crotalus durissus terrificus venom, inhibits macrophage spreading and phagocytic activities. The crotoxin molecule is composed of two subunits, an acidic non-toxic and non-enzymatic polypeptide named crotapotin and a weakly toxic basic phospholipase A(2) (PLA(2)). In the present work, the active subunit responsible for the inhibitory effect of crotoxin on macrophage function was investigated. Peritoneal macrophages harvested from naive rats were used. Crotapotin (2.12, 3.75, or 8.37nM/ml), added for 2h to the medium of peritoneal cell incubation, did not modify the spreading and phagocytic activities of these cells. On the other hand, the PLA(2) (1.43, 2.86, or 6.43nM/ml) subunit caused a significant reduction (30, 33, and 35%, respectively) of the spreading activity. The PLA(2) also inhibited the phagocytosis of opsonised zymosan, opsonised sheep erythrocytes, and Candida albicans, indicating that this inhibitory effect is not dependent on the type of receptor involved in the phagocytosis process. The inhibitory effect of PLA(2) was not due to loss of cell membrane integrity, since macrophage viability was higher than 95%. These findings indicate that the inhibitory effect of crotoxin on macrophage spreading and phagocytic activities is caused by the phospholipase A(2) subunit.

The incorporation and oxidation of arachidonic acid (AA) by rat lymphocytes (LY), the transfer of AA from LY to rat macrophages (Mphi) in co-culture, and the subsequent functional impact on Mphi phagocytosis were investigated. The rate of incorporation of [1-14C]AA by untreated-LY and TG (thioglycolate treated)-LY (TG-LY) was 158 +/- 8 nmol/10(10) LY per h for both untreated-LY and TG-LY. The oxidation of AA was 3.4-fold higher in TG-LY as compared with untreated cells. LY from TG-injected rats had a 2.5-fold increase in the oxidation of palmitic (PA), oleic (OA), and linoleic (LA) acids. After 6 h of incubation, [14C] from AA was distributed mainly into phospholipids. The rate of incorporation into total lipids was 1071 nmol/10(10) cells in untreated-LY and 636 nmol/10(10) cells in TG-LY. [14C]AA was transferred from LY to co-cultured Mphi in substantial amounts (8.7 nmol for untreated and 15 nmol per 10(10) for TG cells). Exogenously added AA, PA, OA, and LA caused a significant reduction of phagocytosis by resident cells. Mphi co-cultured with AA-preloaded LY showed a significant reduction of the phagocytic capacity (about 40% at 35 microM). LY preloaded with PA, LA, and OA also induced a reduction in phagocytic capacity of co-cultured Mphi. TG treatment abolished the AA-induced inhibition of phagocytosis in Mphi co-cultured with TG-LY. Therefore, the transfer of AA between leukocytes is a modulated process and may play an important role in controlling inflammatory and immune response.

Previous work of our group demonstrated that Crotalus durissus terrificus venom has a dual effect on macrophage function: it inhibits spreading and phagocytosis and stimulates hydrogen peroxide and nitric oxide production, antimicrobial activity and glucose and glutamine metabolism of these cells. Crotalid venom also induces analgesia and this effect is mediated by opioid receptors. The involvement of opioidergic mechanism and the determination of the active component responsible for the inhibitory effect of crotalid venom on macrophage function were investigated. The venom reduced the spreading and phagocytic activities of peritoneal macrophages. This effect was observed in vitro, 2 h after incubation of resident peritoneal macrophages with the venom, and in vivo, 2 h after subcutaneous injection of the venom. The inhibition of phagocytosis was not modified by naloxone, an antagonist of opioid receptors. Venom neutralization with crotalid antivenom abolished the inhibitory effect of the venom, indicating that venom toxins are involved in this effect. Crotoxin, the main toxin of crotalid venom, s.c. injected to rats or added to the medium of peritoneal cell incubation, inhibited macrophage function in a similar manner to that observed for crude venom. The present results suggest that crotoxin causes a direct inhibition of macrophage spreading and phagocytic activities and may contribute to the inhibitory effect of crotalid venom on macrophage function.

In the present study, we examined the effect of Crotalus durissus terrificus venom on rat macrophage metabolism and function. Two hours after subcutaneous injection of the venom, peritoneal resident (unstimulated), elicited (thioglycollate-stimulated), and activated Mycobacterium bovis strain bacille Calmette Guérin (BCG) macrophages were collected, and their functional and metabolic parameters were analyzed. The venom inhibited spreading and phagocytosis of macrophages. On the other hand, this treatment increased H(2)O(2) and NO production, candidacidal activity, and the activities of key enzymes of glycolysis and glutaminolysis. We also investigated whether the venom could affect macrophage activation by thioglycollate or BCG. The administration of venom 2 h before injection of thioglycollate and BCG or 2 or 3 days after injection of the thioglycollate or BCG, respectively, did not modify the previous observations. These findings suggest that crotalic venom leads the macrophage to an activated state, with high production of oxygen- and nitrogen-reactive species. This cell activation state does not include inflammatory properties of spreading and phagocytosis.