Arthur J. Chu

Supplemental L‐arginine HCI augments bacterial phagocytosis in human polymorphonuclear leukocytes

That L‐arginine (L‐Arg) augments the host response to acute bacterial sepsis suggests that this amino acid intervenes early in the immune response, perhaps via the nitric oxide synthetase (NOS) pathway. The effect of L‐Arg supplementation on in vitro phagocytosis of fluorescein‐labeled, heat‐killed Staphylococcus aureus by peripheral blood neutrophils (PMNs) from 12 normal human volunteers was studied. Separated PMNs were incubated for 2 h with labeled bacteria, with and without supplemental L‐Arg, D‐arginine, glycine, and/or the NOS inhibitors L‐canavanine, aminoguanidine, or L‐NG‐nitroarginine methyl ester. PMNs were fixed and extracellular fluorescence quenched with crystal violet. By flow cytometry and confocal microscopy, L‐Arg supplementation was shown to result in a highly significant increase in PMN bacterial phagocytosis, the maximal effect being seen with L‐Arg 380 μM and falling off with higher concentrations. This augmentation was completely abrogated by NOS inhibitors in molar excess, but inhibitors alone did not suppress phagocytosis below that of unsupplemented controls. Neither D‐arginine nor glycine affected phagocytosis; the L‐Arg effect was stereospecific and not related to utilization of L‐Arg as an energy source. L‐Arg supplementation significantly enhances bacterial phagocytosis in human neutrophils, perhaps by effects on cytoskeletal phenomena, and this appears to be mediated through NOS activity. Phagocytosis by nonspecific immune cells which intervene early in the response to sepsis is critically important, and beneficial effects of L‐Arg on the clinical course of sepsis may be due at least in part to augmentation of phagocyte function.

Involvement of CD44 and the cytoskeletal linker protein ankyrin in human neutrophil bacterial phagocytosis.

The leukocyte CD44 and CD45 cell surface receptors are associated via the linker proteins ankyrin and fodrin with the cytoskeleton, which itself is important in immune cell functions such as adherence, chemotaxis, and phagocytosis. The effects of rat antihuman CD44 and CD45 monoclonal antibodies on phagocytosis of fluoresceinated heat‐killed Staphylococcus aureus 502A by normal human neutrophils (PMNs) during 2 hr incubation in RPMI‐1640 was studied via flow cytometry and confocal microscopy. Flow cytometry was performed using an excitation wavelength of 488 nm, fluorescence being measured at 515–560 nm on 50,000 PMNs per sample. Confocal microscopy was performed on samples after further incubation with rhodamine‐conjugated antiankyrin. Anti‐CD44 resulted in an increase of 27–31% compared to control (P = 0.004) in the proportion of PMNs fluorescing, an increase of 17–24% (P = 0.001) in mean intracellular fluorescence per PMN, and an increase in total PMN fluorescence of 50–58% compared to control (P < 0.001). In contrast, anti‐CD45 had little effect on phagocytosis. Colchicine (a microtubule‐disrupting agent) enhanced, whereas cytochalasin‐D (a microfilament inhibitor) inhibited bacterial phagocytosis; cytochalasin‐D completely abrogated the effect of anti‐CD44 on this PMN function. Hyaluronic acid augmented phagocytosis by an increment similar to that observed with anti‐CD44. Two‐color flow cytometry and confocal microscopy demonstrated that ankyrin always colocalized with ingested fluorescein isothiocyanate (FITC)‐labeled bacteria. These data strongly suggest that CD44 is involved in bacterial phagocytosis, provide further evidence of CD44 receptor linkage to cytoskeletal elements in human leukocytes, and suggest that ankyrin has a significant role in the transport of phagosomes.

Antagonism by ethanol of endotoxin-induced tissue factor activation in relation to the depressed endotoxin binding to monocyte-like U937 cells.

Our previous study has reported that ethanol (ETOH) partially inhibited the endotoxin (LPS)‐induced tissue factor (TF)‐activation in monocytes including blood peripheral monocytes as well as cultured leukemic U937 and THP‐1 cells. The present study shows a strong correlation (r=0·92; p<0·01) between TF‐activation and depression in LPS binding blocked by ETOH in U937 cells. The antagonism by ETOH of LPS binding was not due to a direct extracellular blockade, since ETOH did not affect the affinity of fluorescein isothiocyanate (FITC)‐LPS or ‐anti CD14 mAb on U937 cells. After U937 cells were treated with 2 per cent (v/v) ETOH for 3 h, LPS binding was however drastically inhibited as shown by immunostaining with FITC‐LPS which was viewed on a confocal laser scanning microscope. The results imply that cellular events of the ETOH effect mediate this inhibition of LPS binding. Anti‐CD14 mAb (UCHM‐1) inhibited LPS binding in a dose‐dependent fashion, revealing a competitive specific binding to the LPS receptor. The results suggest that CD14 plays an important role in the recognition of LPS. FITC‐UCHM‐1 binding was significantly reduced in the cells pretreated with 2 per cent (v/v) ETOH for 3 h, indicating that ETOH modulates the ability to express CD14. CD14 expression was upregulated by priming with LPS which was offset by ETOH. Acetaldehyde, a possible metabolite of ETOH, was tested with no effect on CD14 expression. Taken together, our results show that ETOH downregulates the recognition of LPS, and suggest that the inhibitory action is likely to be mediated by the depression in CD14 expression which was also accompanied by a significantly altered membrane fluidity. Thus, the antagonism by ETOH of the binding of LPS results in a depression in the LPS‐induced TF‐activation.

Fatty acid unsaturation increases expression and capping of murine lymphocyte CD44 and CD45

We studied the effect of incubating murine lymphocytes with cis-unsaturated fatty acids on expression and capping of CD44 and CD45. Lymphocytes were incubated with stearic (18:0) or oleic (18:1 omega-9) acid bound to bovine serum albumin (BSA). After incubation with rat anti-CD44 or anti-CD45 monoclonal antibodies and then with fluorescent-labeled anti-rat antibody, mean fluorescence intensity (FI) was measured by using flow cytometry. Capping was measured after warning and fixation in paraformaldehyde. Steady-state fluorescence anisotropy (rs) was measured after the cells had been incubated with trimethylammoniumdiphenylhexatriene. Incubation with oleic acid, but not stearic acid or BSA alone, was associated with an increase in FI of CD44. Expression of CD45, however, was increased by both stearic and oleic acids to the same degree over BSA controls. CD44 and CD45 capping were both increased by incubation with oleic acid. Rs was decreased in cells incubated with oleic acid, suggesting an increase in membrane fluidity. We conclude that incubation with oleic acid increases expression of CD44 and increases capping of both CD44 and CD45. These findings were confirmed in feeding experiments, in which rs was reduced and CD44 capping increased by polyunsaturated fatty acid diets.

ASSOCIATION OF MURINE SPLENOCYTE CD3 COMPLEX TO THE CYTOSKELETON: ABSENCE OF MODULATION BY EXOGENOUS FATTY ACIDS

The cytoplasmic regions of the CD3 complex are presumably involved in signal transduction following ligand—receptor binding. We investigated the effects of incubating either stearic or oleic acid on the association of murine lymphocyte CD3 complex with the cytoskeleton. Both cytochalasin D, an inhibitor of microfilament formation, and W7, an inhibitor of calmodulin, inhibited capping of CD3. The association of CD3 with the cytoskeleton was confirmed by confocal laser scanning microscopy studies, which showed co‐localization of the cross‐linked CD3 receptors and the membrane attachment proteins ankyrin and fodrin. Although exogenous oleic acid increased plasma membrane fluidity, neither expression nor capping of CD3 receptors was increased. Nonetheless, oleic acid did increase uptake of tritiated thymidine after binding of anti‐CD3 antibodies. Lymphoproliferation was progressively inhibited by both cytochalasin D and W7, confirming the importance of intact cytoskeleton for cellular activation.