We used monoclonal antibodies and flow cytometry to study the expression of the receptors for the complement fragments C3bi (CR3) and C3b (CR1) on human polymorphonuclear neutrophil leukocytes (PMN). Expression of both receptors was minimal on cells stained in anticoagulated whole blood incubated at 0 degree or 37 degrees C. PMN isolated with Percoll density gradients and held at 0 degree C also had only minimal expression of both receptors. With the isolated cells, however, a spontaneous increase in expression of both receptors occurred upon warming to 37 degrees C. This did not represent complete expression of either receptor since additional increments in surface expression could be induced upon stimulation with N-formyl-methionyl-leucyl-phenylalanine or Raji cell supernatant. The increases in complement receptor (CR) expression appeared to be specific since there were no changes in expression of the Fc gamma receptor or beta-2-microglobulin under any of these conditions. The increased CR expression seems to involve translocation from an intracellular pool since it is complete within minutes and is not blocked by puromycin or cycloheximide. These results demonstrate that both CR3 and CR1 expression increase rapidly upon activation of PMN and that isolated cells can be used to study this phenomenon, which may be a critical part of neutrophil function in vivo.

/pdf/human-neutrophils-increase-expression-of-c3bi-as-well-as-c3b-14jw5br1pd.pdf

Human neutrophils increase expression of C3bi as well as C3b receptors upon activation.

Chlorination of endogenous amines by isolated neutrophils. Ammonia-dependent bactericidal, cytotoxic, and cytolytic activities of the chloramines.

Immunologic basis for increased susceptibility of the neonate to infection.

Isolated neutrophilic leukocytes were incubated with primary amines and related nitrogenous compounds. Stimulation of neutrophil oxygen (O2) metabolism with phorbol myristate acetate or opsonized zymosan resulted in production of hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (C1-) to hypochlorous acid (HOC1), and the reaction of HOC1 with the added compounds to yield nitrogen-chlorine (N-C1) derivatives. Formation of N-C1 derivatives of low lipid solubility resulted in accumulation of the derivatives in the extracellular medium. These oxidizing agents were identified and measured on the basis of their absorption spectra and their ability to oxidize 5-thio-2-nitrobenzoic acid to the disulfide form. The yield of N-Cl derivatives was in the order: taurine greater than Tris greater than spermidine greater than spermine greater than glucosamine greater than putrescine greater than guanidinoacetate. Accumulation of N-C1 derivatives was also observed in the absence of added amines, owing to the reaction of HOC1 with endogenous taurine and other amines that were released from the cells into the medium. In the presence of compounds that yield lipophilic N-C1 derivatives, little or no accumulation of oxidizing agents was observed. Instead, these compounds inhibited the accumulation of N-C1 derivatives that was obtained with taurine, and their effect was competitive with taurine. Inhibition was in the order: methylamine greater than ethanolamine greater than phenylethylamine greater than p-toluenesulfonamide greater than ammonia greater than guanidine. Formation of lipophilic N-C1 derivatives also resulted in inhibition of O2 uptake and glucose metabolism. Inhibition was prevented by adding catalase to eliminate H2O2, dapsone to inhibit myeloperoxidase, taurine to compete for reaction with HOC1, or compounds that are rapidly oxidized by HOC1 or N-C1 derivatives, to reduce these oxidizing agents. The results indicate that: (a) formation of N-C1 derivatives that do not penetrate biological membranes can protect leukocytes against the cytotoxicity of HOC1 and lipophilic N-C1 derivatives, and (b) formation of membrane-permeable N-C1 derivatives in the absence of target cells or readily oxidized substances results in oxidative attack by the N-C1 derivatives on leukocyte components and inhibition of leukocyte functions.

/pdf/myeloperoxidase-dependent-effect-of-amines-on-functions-of-4tbk79o8uo.pdf

Myeloperoxidase-dependent effect of amines on functions of isolated neutrophils.

Activation of human neutrophils (PMN) is accompanied by rapid upregulation of CR1, the C3b receptor, and CR3, the iC3b receptor, which also serves as the PMN's major adherence protein. This is necessary for migration and phagocytosis, but the extent of expression of these proteins on PMN at inflammatory sites has not been determined. We used monoclonal antibodies and flow cytometry to assess CR1 and CR3 expression on PMN in bronchoalveolar lavage (BAL) fluid of cystic fibrosis (CF) patients chronically infected with pseudomonas and in sterile joint fluid of arthritis patients. Resting peripheral blood PMN from these patients and normals expressed similar low levels of CR1 and CR3, and the patients' PMN increased CR1 and CR3 expression normally when stimulated in vitro. CR3 expression on CF BAL PMN was 90 +/- 12% of that on the same patient's blood cells stimulated in vitro with FMLP. In contrast, CR1 expression on BAL PMN was only 27 +/- 8% of that on stimulated blood cells. Similar results were obtained for joint PMN. This pattern could be reproduced in vitro by treating FMLP-stimulated blood cells with BAL supernatants or with pseudomonas or PMN elastase. The serine protease inhibitors, PMSF and alpha 1-antitrypsin prevented the lavage supernatant from reducing CR1 expression, while metalloprotease inhibitors had no effect. Treatment of PMN with elastase in vitro decrease their ability to kill opsonized Pseudomonas aeruginosa. These results suggest that PMN at inflammatory sites have maximally upregulated expression of their complement receptors, but that CR1 is then cleaved by proteolysis in situ. Although not related to the basic defect in CF, this may interfere with efficient phagocytosis and contribute to the CF patient's inability to eradicate chronic lung infection.

/pdf/complement-receptor-expression-on-neutrophils-at-an-1u56ot19g1.pdf

Complement receptor expression on neutrophils at an inflammatory site, the Pseudomonas-infected lung in cystic fibrosis

The isolation and functional activity of polymorphonuclear leukocytes and lymphocytes separated from whole blood on a single Percoll density gradient

The requirement for linked HLA-D antigen recognition for the proliferation of antigen-specific T-cell blasts provides a method for testing antigen presentation of human monocytes. We used maternal tetanus toxoid-specific T-cell blasts to show that human newborn monocytes can process and present antigen at least as well as maternal monocytes. These results suggest that human monocytes are relatively more mature at birth than those of newborn rats and mice. A major role for monocyte-macrophage immaturity in limiting the immune responses of human newborns is therefore unlikely.

James T. Kurnick

Papers

The isolation and functional activity of polymorphonuclear leukocytes and lymphocytes separated from whole blood on a single Percoll density gradient

Presentation of antigen by human newborn monocytes to maternal tetanus toxoid-specific T-cell blasts