The objective of this study was to determine the direction of membrane lipid flow in locomoting cells. The plasma membrane of human polymorphonuclear leukocytes was stained with a fluorescent lipid analog dihexadecanoyl indocarbocyanine. A line was photobleached on the cell surface perpendicular to the direction of cell motion. Low-light-level fluorescence microscopy and digital image-processing techniques were used to analyze a series of images taken at short intervals after photobleaching. The bleached line remained visible for about 5 seconds before being erased by diffusional recovery. Examination of fluorescence intensity profiles allowed a comparison to be made between the velocities of line and cell movement. Results indicate that the bleached line moves forward with the same velocity as the cell during locomotion, refuting the retrograde lipid flow model of locomotion. Instead, the plasma membrane lipid appears to move forward according to either the unit movement of membrane or the tank track model of locomotion.

The relative contributions of host resistance and bacterial virulence were analyzed in a mouse model for ascending urinary tract infection. The congenic mouse strains C3H/HeJ and C3H/HeN were used in parallel. They differ in their reactivity to lipopolysaccharide (LPS) and susceptibility to experimental urinary tract infection. C3H/HeJ cells are susceptible to infection and are nonresponders to LPS (Lpsd Lpsd), whereas C3H/HeN cells respond to LPS and are resistant to infection (Lpsn Lpsn). The Escherichia coli pyelonephritis isolate GR-12, serotype O75K5, expressing adhesins specific for globoseries glycolipids (P fimbriae) and for mannosides (type-1 fimbriae), and its derivatives deficient in these factors were used, either singly or in combination, to establish experimental infections. In C3H/HeN mice, the relative persistence of E. coli was inversely proportional to its phagocytosis in vitro. Loss of the O75 and K5 antigens increased the tendency toward hydrophobic interaction, promoted phagocytosis, and reduced persistence in the kidneys. This was not the case in C3H/HeJ mice, in which O75- and K5- serotypes persisted in the same extent as did the parent strain. The total number of bacteria recovered from the kidneys of C3H/HeJ mice was about 1,000-fold higher than the number recovered from kidneys of C3H/HeN mice 24 h after infection. Previous studies have demonstrated a delayed influx of polymorphonuclear leukocytes into the urinary tracts of C3H/HeJ mice. The results are consistent with the hypothesis that phagocyte activation through LPS is a major defense mechanism against E. coli in the kidney, a property in which C3H/HeJ mice are deficient.

The fitness between bacterial adhesins and target cell receptors, determining bacterial adherence to epithelial cells in urinary tract infections, was shown to influence also the interaction with human polymorphonuclear leukocytes (PMNL). Two sets of homogenic strains, constructed to express either, both, or none of the globotetraosylceramide-sensitive (GS) adhesins specific for globoseries glycolipid receptors or the mannose-sensitive (MS) adhesins inhibited by a-methyl mannoside were compared regarding charge, hydrophobicity, and binding to PMNL. The mutants of a hydrophilic pyelonephritis strain required MS adhesins for binding to and activation of the PMNL. Removal of the MS adhesins from the mutant carrying both MS and GS adhesins abolished chemiluminescence and binding. A pronounced chemiluminescence reaction was induced by the hydrophobic strain without GS or MS adhesins. Transformants of this strain expressing the MS adhesin bound to and activated the PMNL. Poor binding and activation were found with mutants and transformants carrying only the GS adhesins. The improved reactivity after coating of the PMNL with the appropriate receptor glycolipid supported the previously reported absence of globoseries glycolipids in those cells as the reason for the refractoriness to bacteria with GS adhesins. The mechanism of binding, which improves epithelial cell adhesion, may prevent binding to PMNL, thus improving the survival of Escherichia coli in the kidney.

The stimulation of neutrophils by formylmethionyl-leucyl-phenylalanine results in a bimodal luminol-dependent chemiluminescence pattern. We observed, however, only a single peak chemiluminescence pattern in the response to the peptide when we used lucigenin as an amplifying substance. We suggest that lucigenin, the larger molecule, (510 daltons; luminol is 177 daltons) only exerts an extracellular effect.

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