Effect of low neutral endopeptidase expression on response to fMLP.

The effects of the low neutral endopeptidase (24.11/CD10) exhibited by cord blood neutrophils on response to the peptide mediator of cell function f-met-leu-phe (fMLP) were investigated. Oxidative radical release (superoxide and hydrogen peroxide) and chemotactic responses to fMLP were determined and compared to the responses of normal adult neutrophils. The effect of fMLP on CD10 expression as measured by flow cytometry also was evaluated. The data show that cord blood neutrophils produce increased amounts of O2- and H2O2 largely because of a prolonged reaction time to fMLP. In addition, adult polymorphonuclear neutrophil leukocytes increase the intensity of their expression of CD10 following fMLP stimulation, whereas cord blood CD10 expression does not change. Evaluation of chemotaxis demonstrated that cord blood neutrophils exhibited a shift in the fMLP dose-response relationship showing relatively better chemotaxis to lower concentrations. In support of this observation, the inhibition of endopeptidase on adult polymorphonuclear neutrophils leukocytes by phosphoramidon was associated with an augmentation of chemotaxis to 10(-9) and 10(-10) mol/L fMLP. These studies demonstrate that cord blood and adult neutrophils respond differently to fMLP and suggest that membrane endopeptidase plays a role in the observed response patterns. The low level of expression of CD10 on cord blood neutrophils and the failure to increase its expression after fMLP stimulation suggests that adult neutrophils have preformed intracellular CD10 that is not present in the newborn. We propose that the lack of endopeptidase on cord blood neutrophils together with other known features of immaturity may play a role in the overall compromised host defense exhibited by the newborn.

Inhibition of chemotaxis Ng-monomethyl-L-arginine: a role for cyclic GMP.

The metabolism of L-arginine to nitric oxide (NO) has been shown to be important for the effector functions of many cell types, including polymorphonuclear (PMN) leukocytes. Its effect appears to be mediated at least in part by NO stimulation of soluble guanylate cyclase. We evaluated the role of this pathway in two PMN effector functions: cell movement and microbial killing, using the competitive inhibitor of L-arginine conversion to NO, NG-monomethyl-L-arginine (NMA). We also evaluated the effect of additional L-arginine and dibutyryl cyclic guanosine monophosphate (cGMP) on any NMA-associated changes. Human peripheral blood neutrophils were used and the cells were incubated with and without NMA. Chemotaxis was evaluated using a 48-well micro-Boyden chamber. Microbial killing was evaluated using S aureus strains D2C and 502A. These studies demonstrated that chemotaxis to formyl-methionyl-leucyl-phenylalanine was markedly inhibited in NMA-treated cells. This inhibition could be overcome if L-arginine or dibutyryl cGMP were added with the NMA. In contrast, microbial killing of S aureus was unaffected by NMA. These observations support the hypothesis that the L-arginine metabolism to NO and its effect on the cGMP level may be important for the dynamic changes required for neutrophil chemotaxis.

Inhibition of cell movement and associated changes by hexachlorocyclohexanes due to unregulated intracellular calcium increases.

Hexachlorocyclohexanes (HCCHs) are potent stimulators of polymorphonuclear leukocyte (PMN) oxidative metabolism and of mobilization of calcium from intracellular stores. It was of interest, therefore, to evaluate the effect of HCCHs on PMN orientation and chemotaxis and to determine their effectiveness as chemotaxins. Chemotaxis was evaluated using micro-Boyden chambers, f-actin was quantitated by nitrobenzoxadiazole (NBD)-phallacidin fluorescence, and microtubules were quantitated by observing the concanavalin A (Con A) capping phenomenon. We also evaluated changes in intracellular calcium [Ca2+]i using quin 2 fluorescence. We found that the HCCH isomers were not chemotaxins and that the HCCH isomers that stimulated O2- formation (delta and gamma HCCH) inhibited chemotaxis. This effect was associated with inhibition of orientation. In addition, we found extensive inhibition of both f-actin and Con A cap formation. These effects of HCCH on cell function were associated with marked increases of [Ca2+]i. This work suggests that non-receptor-mediated increases of [Ca2+]i associated with HCCH have divergent effects on cell function and suggests that physiologic responses of PMNs requiring cytoskeletal alterations, such as chemotaxis, depend on the controlled responses of receptor-mediated stimulation.

Transient chemotactic defect in a child with elevated IgE.

Leukocyte function was studied in a child with elevated IgE and many infections. At age 7 months, chemotaxis was decreased, but improved first toward formylated peptides and finally toward complement. Polarization and nitroblue tetrazolium reduction also were transiently impaired. Functional and clinical improvements were concomitant although IgE remained elevated.

Monoclonal antibodies to CALLA do not alter polymorphonuclear functions.

The common acute lymphoblastic leukemia antigen (CALLA) is present on the malignant cells of most patients with acute lymphoblastic leukemia (ALL). Monoclonal antibodies (MoAbs) to CALLA have been useful for differentiating lymphoblastic from nonlymphoblastic leukemias as well as for serotherapy in ALL. Since this MoAb also reacts with polymorphonuclear (PMN) leukocytes, it was of interest to determine whether or not MoAbs to CALLA affected PMN function. We therefore evaluated four MoAbs to CALLA for their effect on PMN function. Two of the MoAbs were IgG and two were IgM. Chemotaxis was studied using Micro-Boyden chambers. Intraleukocyte bacterial killing was studied using Staph:PMN of 2:1 and 10:1, and metabolic activation was evaluated by hexose monophosphate shunt activity. The results showed that these MoAbs to CALLA did not impair the parameters of PMN function studied. These findings have relevance to the usefulness of MoAbs to CALLA for serotherapy and also as a probe for understanding the surface molecule properties that have a bearing on PMN host defense functions.

Increased plasma chemoattractant in Sweet's syndrome.

The neutrophil function and plasma leukotactic activity of a patient with Sweet's syndrome and cystonodular acne were evaluated during a 2 1/2-year period. These studies demonstrated that chemotaxis was frequently slightly increased, especially during an exacerbation of Sweet's syndrome, but showed some decrease during isotretinoin therapy. Other functions, such as phagocytosis, metabolic activation, and bacterial killing, also were slightly increased. In addition, the patient's serum contained a heat-stable, nonlipid chemoattractant that was present at all times except during a course of isotretinoin. Although his symptoms responded to aspirin, the plasma continued to show this chemoattractant. These findings are consistent with the hypothesis that excess chemoattractant in Sweet's syndrome attracts neutrophils, which then mediate an inflammatory response. In addition, aspirin may be used to control Sweet's syndrome symptoms, although it does not suppress the plasma chemoattractant.