MANNOSE-SPECIFIC INTERCELLULAR AGGREGATION OF RAT THYMOCYTES AND ESCHERICHIA COLI CELLS TRIGGERED BY TEMPERATURE

D-mannose was shown to induce disaggregation of rat thymocytes and E. coli ЛВ1157 cells at any stage of the process. Mannose-specific aggregation of thymocytes and E. coli was inhibited by heating as opposed to concanavalin A-induced thymocyte aggre­ gation. The process was completely reversible at the rapid temperature changes, indi­ cating its membrane not intracellular mechanism. Taken together, the results demonstra­ te that mannose-specific intercellular contacts can be affected differently by temperature.


Introduction.
Results of recent research emphasize that a notable part of intercellular contacts are based on the protein-carbohydrate interaction [1,2].Bacterial cells can effectively interact with animal cells in adhe sion and intercellular aggregation reactions [3,4].The mechanisms of these processes are being studied intensively since they are the basis of the development of various bacterial infections [5].It has been shown that E. coli cells bearing type 1 fimbrial lectin bind to mannose-eontaining glycoligands on the surface of macrophages and neutrophils [6].Recently, we have found that E. coli AB1157 cells also induce aggregati on of rat thymocytes, while heat-inactivated bacterial cells lose this ac tivity [7].Earlier, heating was shown to affect lectin-dependent interac tions in cellular and model (liposomes) systems [8,9].The mannose-spe cific intercellular aggregation of rat thymocytes and E. coli may be used to model lectin-carbohydrate contacts between animal and bacterial ceils in suspensions, because, in comparison with phagocytes, thymocytes have little unspecific adhesion to a majority of surfaces.
In the present study we investigated the effect of the temperature provoking structural and morphological changes in the cell plasma memb rane on the rat thymocyte aggregation induced by E. coli AB1157 and mannose-binding lectin concanavalin A (Con-A).Our results show that in contrast to Con-A-induced aggregation, the mannose-specific intercel lular aggregation of rat thymocytes and E. coli is triggered by the rapid temperature change and occurs at the another temperature range.
Experimental.D-mannose and Con-A were obtained from «Sigma» (USA).
E. coli AB1157 cells were cultured by the standard method [10].Bri efly, the bacteria were grown overnight at 37 °C in a beef-extract broth and washed two times with PBS before use.
The aggregation of rat thymocytes induced by E. coli as well as by Con-A was measured by changes in light transmission at 590 nm of cell suspensions [7], which were kept at a constant temperature and wilh continuous stirring.
Results and discussion.The rat thymocytes aggregated in the pre sence of E. coli AB1157 at room temperature (fig.1).The order in which cells were added played no role in the results, the intercellular aggrega tes were microscopically visible.To test the stability of cell aggregates the gaptenic sugar D-mannose was added at various time points of the aggregation process.Independent from addition time, D-mannose in con centration of 45 mM elicited the complete disaggregation of cells (fig.1).

Fig. I.
Aggregation of rat thymocytes by E. coli.E. coli cells (l.5-10 8 /ml) were added at time zero to thymocyte suspension (7-l0 6 /ml) in PBS at 20 °C; D-mannose (45 mM) was added after the bacteria as indicated by the arrows.The traces are representative of at least three experiments Fig. 2. Effect of temperature on rat thymocyte aggregation induced by E. coli and Con-A.E. coli cells (l.3-10 8 /ml) or Con-A (50 pig/ml) were added to rat thymocyte suspension (4.5-l0 6 /ml) in PBS kept at the indicated temperature.Aggregation rate was calculated as an incline of tangent to bend point of trace.The illustrated data are re presentative of three experiments Earlier, other sugars (D-glucose, D-galactose, lactose, D-maltose, L-rhamnose et al.) were found to be inactive to affect the intercellular aggrega tion [7].The high carbohydrate specificity of this reaction prompted us to compare it with another mannose-specific aggregation of rat thymo cytes induced by Con-A.
Fig. 2 illustrates the effect of temperature on the rat thymocyte ag gregation induced by E. coli in comparison with those induced by mannosc-binding lectin Con-A.It was observed that the rate of thymocyte and E. coli intercellular aggregation decreased with the temperature increa se.On the contrary, heating raised the rate of Con-A-induced thymocyte aggregation (fig.2).Thus, mannose-specific contacts of cell surfaces formed by bacterial and plant lectins display different sensitivity to temperature.
Since at 50 °C rat thymocytes and E. coli did not interact (fig.2), we hypothesized that high temperature might destroy intercellular con tacts due to the reversible heat-induced structural changes of respective plasma membrane components.This supposition was proven by special experiments, when the temperature of samples was changed rapidly (for 15-25 s) during the process of aggregation.Indeed, the intercellular ag gregation of rat thymocytes and E. coli proceeding at 16 °C was stopped with the rapid jump in the temperature of the sample to 50 °C, thus the cell aggregation process was replaced by disaggregation (fig.3).Further more, it was reversible with the following rapid decrease in temperature to 16 °C (fig.3).At the same time, Con-A-induced aggregation of thy mocytes could be stopped by rapid fall in temperature without cell dis aggregation (not illustrated).
The results of the study reported here show that intercellular con tacts between E. coli AB1157 and rat thymocytes are based on the man nose-specific interaction of respective surface structures.Apparently, the type 1 fimbrial lectin of bacterial cells takes part in this reaction as well as in process of lectinophagocytosis of the bacteria by phagocytes [6].
Since the lection is expressed by many virulent strains of E coli [11,12] the possibility of forming intercellular contacts of bacterial and lym phoid cells may be significant for the development of the local immune response.