Cyclic nucleotide changes induced in human leukocytes by a product of axenically grown Entamoeba histolytica that inhibits human monocyte locomotion.

Pulse exposure of human mononuclear phagocytes to the monocyte locomotion-inhibitory factor produced by Entamoeba histolytica (i.e., the 369- to 765-Da chromatographic fraction obtained from the supernatant fluid of axenically grown E. histolytica) led to a swift increase in the intracellular concentration of adenosine 3':5' cyclic monophosphate (cAMP). A weaker response was observed in human polymorphonuclear leukocytes, the locomotion of which, however, is not inhibited by this amebic factor. The same chromatographic fraction obtained from the axenic medium control lacked this effect, at least upon mononuclear phagocytes. On the other hand, both the monocyte locomotion-inhibitory factor and the axenic medium control, possibly through shared cultured medium components, induced comparable increases in guanosine 3':5' cyclic monophosphate (cGMP) in human mononuclear phagocytes and in polymorphonuclear leukocytes, thus suggesting that the latter-nucleotide is not critical for the leukotactic inhibitory phenomenon. Our results suggest that like other leukotactic inhibitors, the monocyte locomotion-inhibitory factor produced by E. histolytica operates through modulations of intracellular cAMP.

Further physicochemical characterization of the monocyte locomotion inhibition factor (MLIF) produced by Entamoeba histolytica.

Ultrafiltration, gel-sieve chromatography and HPLC were used to purify MLIF. This material was HCl-hydrolyzed and the amino acids analyzed by HPLC-ortho-ophthaldialdehyde. Two hydrophobic non-polar (ile,leu), two uncharged polar (thr,tyr) and two positively charged (basic) (his, arg) amino acids were found in clear excess to their concentration in AMC and thus may intervene in the composition of MLIF.

Effect of the monocyte locomotion inhibitory factor (MLIF) produced by Entamoeba histolytica upon the respiratory burst of human leukocytes.

In addition to inhibiting the locomotion of human MP, MLIF appears capable of inhibiting the respiratory burst (measured by chemiluminescence) of MP and of PMN as well. The effect on the latter cells may or may not be relevant in the host-E. histolytica interaction, as PMN have been found to be notoriously inefficient in dealing with amebas and, foremost, do not use oxidative mechanisms in dealing with the parasite. On the other hand, the inhibitory effect on MP may represent a true evasion mechanism inasmuch as activated MP are capable of destroying virulent amebas, and do so by both oxidative and non-oxidative mechanisms.

The role of mannose in the receptor of the monocyte locomotion inhibitory factor produced by Entamoeba histolytica.

The effect of the monocyte locomotion inhibitory factor (MLIF) produced by Entamoeba histolytica is diminished, if not cancelled, when human monocytes are pre-exposed to concanavalin A or sodium periodate, respectively, but not when MLIF is pretreated with sodium periodate. When the MLIF-inhibited monocyte locomotion assays were performed in the presence of 12 different carbohydrates, only the runs containing D-mannose, 4-O-beta-galactosyl-mannoside or mannan revealed a significant reduction in the inhibitory effect. Finally, the MLIF activity was virtually absorbed out with mannan-coupled Sepharose 4B beads. This suggests that D-mannose constitutes an essential part of the receptor for MLIF on the human monocyte membrane.

[Monocyte locomotion inhibiting factor (MLIF) produced by E. histolytica induces an increase of cAMP in human monocytes]. / El factor inhibidor de la locomoción de monocitos (FILM) producido por E. histolytica induce aumento del AMPc en los monocitos humanos.

The supernatant fluid of axenically grown E. histolytica contains a factor (MLIF) which inhibits the locomotion of human monocytes (including chemotaxis) without affecting that of human polymorphonuclear leucocytes. Locomotion, like other cellular functions, is modulated by changes in intracellular cAMP and cGMP. The consensus--with some exceptions--is that while rises in cGMP accompany locomotion, an increase in cAMP (without a concomitant fall in -cGMP) occurs with inhibition of cellular movement. We measured by radioimmunoassay the cAMP concentration of human monocytes exposed to inhibitory concentrations of MLIF. A significant (p less than 0.005) rise in monocyte cAMP was found, comparable to that observed with the use of forskolin, a well known cAMP stimulator. The control studies using plain axenic medium, not only failed to reveal any rise in cAMP but disclosed a small, yet not significant drop in intracellular cAMP. These results suggest that MLIF (like other locomotion inhibitors, i.e. prostaglandins E1, A1 and isoproterenol) produces a significant increase in intracellular monocyte cAMP. This modification in intracellular signals may contribute to the inhibition in monocyte locomotion, an event during which an increase in pericentriolar microtubules has also been observed.