P. C. J. Leijh

Requirement of extracellular complement and immunoglobulin for intracellular killing of micro-organisms by human monocytes.

The role of serum factors in the intracellular killing of bacteria by monocytes was studied on the basis of an assay independent of phagocytosis. After 3 min of phagocytosis of preopsonized bacteria and removal of noningested bacteria, the monocytes containing bacteria are reincubated for various periods and the number of unkilled bacteria is determined by a microbiological method after lysis of the cells. Evidence that this assay measures the killing of ingested bacteria was provided by scanning electron microscopy, lysostaphin treatment, and the effect on the rate of intracellular killing of inactivated serum lacking specific opsonic activity. Intracellular killing of Staphylococcus aureaus, S. epidermidis, and Escherichia coli by human monocytes does not occur or is low in the absence of serum, and maximal killing is only reached when fresh serum is present; intermediate values are obtained in the presence of heat-inactivated serum. These findings indicate that complement stimulates intracellular killing. Isolated heterogeneous immunoglobulin (Ig)G, pFc fragments of heterogeneous IgG, and both IgG1 and IgG3 stimulate intracellular killing of S. aureaus by monocytes to the same degree as heat-inactivated serum. Sphingomyelinase, which decreases the number of Fc receptors, and neuraminidase, which increases these receptors, respectively, decreased and increased the intracellular killing, whereas anti-monocyte serum completely abolished the stimulation of intracellular killing by inactivated serum. These results prove that interaction of the Fc receptor with the Fc part of IgG is required for the intracellular killing. Inhibition of the activation of complement components via the alternative pathway gave a considerable reduction in the intracellular killing of S. aureaus; impairment of the activation via the classical pathway had no effect. The addition of complement components to heat-inactivated serum showed that intracellular killing is maximal only when C3b is generated. Reduction of the number of C3b receptors in the membrane by trypsin or pronase decreased intracellular killing in the presence of fresh serum; anti-monocyte serum completely abolished the stimulation of intracellular killing by fresh serum. These results lead to the conclusion that intracellular killing is also dependent on the interaction between C3b and its receptor in the membrane.

Contribution of granulocytes and monocytes to resistance against experimental disseminatedCandida albicans infection

The aim of the present study was to investigate the contribution of granulocytes and monocytes to resistance against an acute systemic candidal infection in mice. To this end granulocytopenia and monocytopenia were induced by irradiation or treatment with cyclophosphamide, and monocytopenia was obtained by treatment with VP-16. After intravenous injection of 1×104Candida albicans into mice irradiated with 8 GY, the number ofCandida albicans cultured from the kidneys, expressed as the geometric mean of the number of CFU/g tissue, was 5.4×104, 7.1×106 and 5.8×107 on days 1, 3 and 5 of infection respectively (p<0.001 compared to normal mice). The number ofCandida albicans cultured from the liver and spleen was also significantly higher for irradiated animals than for normal mice (p<0.001). For cyclophosphamide-treated mice the number of organisms in the kidney (1.7×104 CFU/g on day 1, 1.9×106 on day 3 and 3.8×106 on day 5 of infection) and spleen was significantly higher (p at least <0.02) than for nomal mice after injection of 1×103Candida albicans. Monocytopenia induced by VP-16 did not result in an increase in the number ofCandida albicans cultured from the kidney or spleen after infection. From these studies it is concluded that granulocytes and not monocytes or exudate macrophages play an important role in resistance againstCandida albicans during the first five days of a systemic infection.

Quantitative immunocytochemical characterization of mononuclear phagocytes: I. Monoblasts, promonocytes, monocytes, and peritoneal and alveolar macrophages☆

The purpose of the present study was to compare the phenotype of tissue macrophages with that of their precursors in the bone marrow and blood. The phenotype was determined on the basis of the quantitative binding of monoclonal antibodies to cell-surface antigens (antigen F4/80, complement receptor III, Fc receptor II, Ia antigen, common leukocyte antigen, and Mac-2 and Mac-3 antigens) on individual mononuclear phagocytes. Monoclonal antibody binding to cells, detected by the biotin-avidin immunoperoxidase procedure, was quantitated by cytophotometric determination of the amount of enzyme reaction product on cells. The results of this quantitation are expressed as the median of the specific absorbance per unit of cell-surface area (0.25 micron2) and per cell. Shortly after collection of the mononuclear phagocytes, binding of all monoclonal antibodies except those directed against the common leukocyte and Mac-2 antigens to peritoneal macrophages was enhanced compared with binding to blood monocytes; for alveolar macrophages we found reduced binding of monoclonal antibodies F4/80 and M1/70 (complement receptor III) and enhanced binding of monoclonal antibodies with specificity for the common leukocyte antigen and Mac-2 and Mac-3 antigens. The results obtained with cultured mononuclear phagocytes show that during the development from monoblast to tissue macrophages, monoclonal antibody binding to the various types of mononuclear phagocyte, expressed per unit of cell-surface area, was not significantly altered except that of M3/38 (Mac-2 antigen) to peritoneal macrophages and that of F4/80 and M1/70 (complement receptor III) to alveolar macrophages. Expressed on a per cell basis, the results show an increase in the binding of all monoclonal antibodies except those directed against the Fc receptor II and Mac-3 antigen during the development from promonocytes to peritoneal macrophages; binding of most monoclonal antibodies to alveolar macrophages was considerably lower than that to blood monocytes. It is concluded that the expression of the various cell-surface antigens alters during mononuclear phagocyte differentiation. The expression changed also during culture, although distinct patterns of alteration could not be distinguished.

Complete and partial deficiencies of complement factor D in a Dutch family.

A young man suffering from recurrent Neisseria infections was shown to lack detectable serum complement factor D hemolytic activity. Addition to the patients serum of purified factor D to a final concentration of 1 microgram/ml resulted in full restoration of the activity of the alternative pathway. Using an enzyme-linked immunosorbent assay, it was shown that the patients serum did not contain measurable amounts of factor D antigen either. The sister, the father, as well as the parents of the mother had factor D levels within the normal range, and the factor D level of the mother was decreased. The capacity of the patients serum, at concentrations up to 5%, to promote phagocytosis of Escherichia coli by normal human granulocytes was low when compared to normal serum. Substitution of the patients serum with purified factor D resulted in a full restoration of opsonic activity. This study describes the first complete deficiency of factor D, and demonstrates its possible relation to recurrent Neisseria infections.

Effect of irradiation cyclophosphamide, and etoposide (VP-16) on number of peripheral blood and peritoneal leukocytes in mice under normal conditions and during acute inflammatory reaction

In order to develop a suitable model for studying the role of granulocytes and monocytes in resistance against pathogenic microorganisms, we investigated the effect of irradiation and cytostatic treatment (cyclophosphamide and VP-16) on the number of both peripheral blood and peritoneal leukocytes in male Swiss mice. Irradiation and cyclophosphamide treatment severely decreased the number of both granulocytes and monocytes in peripheral blood, whereas VP-16 only lowered the number of blood monocytes to a significant degree and had little effect on the number of blood granulocytes or lymphocytes. When normal mice were injected intraperitoneally with newborn calf serum (NBCS) the number of peritoneal granulocytes rose about 100-fold within 6 h. In irradiated and cyclophosphamide-treated mice, this influx of granulocytes into the peritoneal cavity was virtually eliminated, as was the concomitant increase in the number of blood granulocytes; in VP-16-treated mice, on the other hand, the number of peripheral blood and peritoneal granulocytes increased to the same degree as in normal mice. An increase in the number of peripheral blood monocytes and peritoneal macrophages occurred 24–48 h after injection of NBCS in normal mice. This increase was significantly impaired by irradiation as well as by treatment with cyclophosphamide or VP-16.

Quantitative study of enzyme immunocytochemical reactions performed with enzyme conjugates immobilized on nitrocellulose

SummaryThe nitrocellulose binding assay was used for quantitative studies on the cytochemical reactions for the three enzymes most frequently used in immunocytochemistry. The results show a linear relationship between the amount of enzyme immobilized on nitrocellulose and the amount of the enzyme reaction product. The similar course of the formation of the reaction product after DAB/H2O2 staining for peroxidase immobilized on nitrocellulose and for immunoperoxidase labeled cells indicates a linear relationship between the amount of enzyme-coupled antibodies bound to cells and the amount of enzyme reaction product. Furthermore, a mild acid treatment for the abolition of endogeneus peroxidase activity in tissues and cells applicable to immunoperoxidase staining procedures is proposed.

Intracellular K+, Na+ and Cl− concentrations and membrane potential in human moncytes

The relationship between the resting membrane potential and the intracellular ionic concentrations in human monocytes was investigated. Cell volume, cell water content, and amount of intracellular K+, Na+, and Cl- were measured to determine the intracellular concentrations of K+ (Ki), Na+ (Nai) and Cl- (Cli) of monocytes, and of lymphocytes and neutrophils. Values found for monocytes were similar to those for neutrophils, i.e., cell volumes were 346 and 345 micron3, respectively, cell water content 78%, and Ki, 128 and 125, Nai, 24 and 26, and Cli, 102 and 103 mmol/l cell water, respectively. Lymphocytes, however, had different values: 181 micron3 cell volume, 77% cell water content, and for Ki, Nai, and Cli, 165, 37, and 91 mmol/l cell water, respectively. The resting membrane potential of cultured human monocytes (range -30 to -40 mV), determined by measurement of the peak potential occurring within the first milliseconds after microelectrode entry, was most dependent on extracellular K+, followed by Cl-, and Na+. The membrane permeability ratio of Cl- to K+ was estimated by use of the constant field equation to be 0.23 (range 0.22 to 0.30).

A cytophotometric method to quantitate the binding of monoclonal antibodies to individual cells.

A cytophotometric technique to quantitate binding of (monoclonal) antibodies to individual cells is described. This method is based on the detection of cell-surface antigens by immunoperoxidase staining procedures. The amount of reaction product of the peroxidase reaction with diaminobenzidine/H2O2 was quantitated by computerized scanning cytophotometry. The incubation period of peroxidase with the substrate required for an optimal cytophotometric determination of the reaction product proved to be 60 min. The reproducibility of individual absorbance measurements, the day-to-day reproducibility of the quantitation of monoclonal antibody binding, and the specificity and sensitivity of the detection of defined monoclonal antibodies, established the reliability of the present method. The sensitivity of the indirect immunoperoxidase procedure could be enhanced by using a biotin-avidin-peroxidase staining procedure.

Kinetics of Phagocytosis and Intracellular Killing of Staphytococcus aureus and Escherichia coli by Human Monocytes

Tie kinetic patterns of the phagocytosis and intracellular killing of Staphytococcus and Echerichia coli by monocytes were investigated separately to acquire more insight into the total process, i.e. from the ingestion to the death of the micro‐organisms. Phagocytosis proved to be dependent on: (1) both the bacteria‐to‐monocyte ratio and the monocyte concentration; a concentration of at least 5 × 105 monocytes/ml proved necessary for the measurement of ingestion, whereas the rate of ingestion was found to be proportional to the number of extracellular bacteria until a maximum rate is reached, (2) the serum concentration in the incubation medium, which influenced both the rate of phagocytosis and the maximum number of bacteria taken up by one monocyte, and (3) the temperature, the highest rate of phagocytosis being reached at 37–41°C The intracellular killing proved to be dependent on: (1) the number of bacteria ingested; the rate of killing was proportional to the number of ingested bacteria until a maximum rate was reached; (2) the temperature, since a maximum rate of killing is only reached at 37–41°C: at tower and higher temperatures the rate of killing is lower, in the latter case due to inactivation of extracellular stimuli. These separate data on the ingestion and killing processes made it possible to compute the theoretical numbers of extracellular, viable intracellular, and total intracellular bacteria for a model system consisting of 5×106 monocytes, 5×106 bacteria, and 10% serum. These calculated values are in agreement with the experimental data.

A micromethod for the separate evaluation of phagocytosis and intracellular killing of Staaphylococcus aureus by human monocytes and granulocytes

A micro-assay has been developed for the separate evaluation of phagocytosis and intracellular killing of Staphylococcus aureus by human monocytes and granulocytes. The minimal number of phagocytes required for the investigation of these functional activities of phagocytic cells has been established by performing phagocytosis and intracellular killing experiments at various cell concentrations, bacteria-to-cell ratios, and volumes. The results of these experiments revealed that phagocytosis can be measured in a reliable way, at bacteria-to-cell ratios of 5:1 and 1:1 (cell concentration 5 x 10(6)/ml), in a suspension of 200 microliters. The rate of intracellular killing by monocytes and granulocytes can also be measured with a total phagocytic suspension of 200 microliters and a cell concentration of 5 x 10(6)/ml. From these results it can be concluded that for an independent determination of the phagocytosis and intracellular killing of micro-organisms, 400 microliters of 5 x 10(6) phagocytes/ml is required, i.e., a total of 2 x 10(6) phagocytes. This number of granulocytes can be obtained from 1-2 ml of blood; for monocytes 4-8 ml of blood is required.