Cell surface immunoglobulin. 3. Isolation and characterization of immunoglobulin from nonsecretory human lymphoid cells.

Cells from an established line of Burkitt lymphoma (Daudi) were enzymatically radioiodinated, and labeled Ig from the cell surface was isolated and studied. Subcellular fractionation of labeled cells confirmed that intracellular proteins from the cytoplasm are not iodinated by this method. Radioactive Ig was identified as monomeric (8S) IgM, and an average of 10(5) Ig molecules was found per cell. Ig molecules could be released from the plasma membrane by detergent lysis under nonreducing conditions indicating that attachment of Ig to the plasma membrane occurs via noncovalent interactions. The ratio of micro/L radioactivity in surface Ig was the same as that of total cellular Ig radioiodinated in solution suggesting that a large portion of the Fc fragment is not buried within the membrane. In contrast to the results obtained with cell surface Ig, most intracellular Ig was found as "free" micro- and L chains regardless of whether lysates were labeled with (125)I or cells were labeled with leucine-(3)H. The results indicate that only a small percentage of the total Ig of Daudi cells is associated with the cell surface and suggest that covalent assembly of Ig occurs at or near the time that the molecule becomes part of the plasma membrane. Similarities between cell surface Ig on normal splenic lymphocytes and Daudi cells suggest that the latter is a neoplasm of bone marrow-derived lymphocytes.

Poly I:C accelerates development of diabetes mellitus in diabetes-prone BB rat.

We developed a new experimental model of accelerated diabetes mellitus in the genetically susceptible diabetes-prone BB rat with the administration of the IFN-alpha inducer poly I:C. With this model, there was both an increased incidence and accelerated onset of insulin-dependent-diabetes in poly I:C-treated animals compared with saline-treated controls. All twelve rats administered poly I:C (5 micrograms/gm body weight 3 times/wk) developed diabetes by 57 days of age (100%) compared with 1 of 27 (3.7%) saline-treated controls. Furthermore, the development of diabetes was accelerated in the poly I:C-treated group (mean age +/- SE at onset 52.8 +/- 0.58 days) compared with saline-treated controls (89.3 +/- 2.4 days, P less than 0.01). Additionally, poly I:C-treated rats had higher mean serum IFN-alpha levels than saline-treated rats at weeks 2 and 3 of treatment (210 vs. 27 and 183 vs. 25 U/ml, respectively, P less than 0.001). Poly I:C treatment of 5 Wistar rats, the parental strain, which is not susceptible to diabetes, did not result in insulitis, diabetes, or hyperglycemia. The histopathologic findings of insulitis and decreased immunoreactive islet insulin in poly I:C-accelerated diabetic BB rats and in BB rats with spontaneous diabetes suggest a similar pathophysiology.

Poly I:C induction of alpha-interferon in the diabetes-prone BB and normal Wistar rats. Dose-response relationships.

Although the administration of a fixed dose of the alpha-interferon (alpha-IFN) inducer, polyinosinic polycytidilic acid (poly I:C), accelerates the development of diabetes in DP-BB rats, no reports have characterized the dose-response relationship of poly I:C with serum alpha-IFN levels and the development of diabetes. This study examines the dose-response relationships of poly I:C with the induction of serum alpha-IFN and the development of diabetes in DP-BB and normal Wistar rats. Also tested in this study is the hypothesis that the lack of development of diabetes in poly I:C-treated normal Wistar rats is attributable to a deficient alpha-IFN response. Using poly I:C doses of 0.5, 1.5, 5, and 10 micrograms/g body weight, a direct dose-response relationship was observed in DP-BB rats with the serum alpha-IFN response. Moreover, all doses of poly I:C accelerated the onset of diabetes in BB rats. Serum alpha-IFN levels inversely correlated with time of onset of diabetes (P < 0.01). Also, BB rats with higher levels of serum alpha-IFN were associated with earlier onset of diabetes (P < 0.001). Poly I:C-induced serum alpha-IFN levels were significantly lower in diabetic than in nondiabetic BB rats. In normal Wistar rats, although all doses of poly I:C significantly increased serum alpha-IFN levels, diabetes was not induced. The results of this study indicate that poly I:C administration elevates serum alpha-IFN and accelerates the development of diabetes in BB rats at even very low doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Gender-related variations and interaction of human neutrophil cyclooxygenase and oxidative burst metabolites.

Gender-related variations in human neutrophil membrane bound oxidative metabolism were evaluated employing the calcium ionophore A23187. These included the measurement of cyclooxygenase metabolites of arachidonate by specific RIA determination of thromboxane B2 (TxB2), prostaglandin E2 (PGE2), and 6-Keto PGF1 (6KPGF) as well as the initiation of the oxidative burst by the quantitative evaluation of superoxide (O-2) reduction of nitroblue tetrazolium (NBT). Neutrophils from women generated 30% less TxB2 and PGE2 than those obtained from men. In contrast, the neutrophils from women demonstrated relatively higher O-2 production with a cyclic pattern of both TxB2 and O-2 which correlated with their menstrual cycle. The elevated O-2 generation appeared to inversely correlate with TxB2 production. Further, introduction of an intracellular oxygen centered radical (OCR) scavenger, sodium benzoate, for the hydroxyl (.OH) radical was observed to affect cyclooxygenase metabolism in a dose-response manner. At higher concentrations of sodium benzoate, i.e., 10(-2) M, TxB2 production was inhibited; in contrast, 10(-3) M sodium benzoate enhanced neutrophil TxB2 generation which was particularly marked during times of increased oxidative burst activity, i.e. O-2 production. We conclude that the decreased production of cyclooxygenase metabolites observed in neutrophils from women in part derive from an increased oxidative burst activity. This suggests that a regulatory mechanism may exist between the neutrophil membrane bound oxidative system(s) involving oxygen centered radicals generated during both the oxidative burst and prostaglandin cyclic endoperoxide reduction. Further, these gender-related differences may be partially attributable to variations in circulating endogenous sex steroids.

Developmental aspects of pulmonary defenses in children.

It is now generally accepted that maturational immaturity of lung defense system(s) exists in the young infant and child which, together with other maturational deficiencies of immunologic systems, contribute to their undue susceptibility to infections, many of which are associated with pulmonary infections. The alveolar macrophage (AM), of central importance to lung defense, has been studied extensively in the neonatal rabbit by our group and in the human, by others. Collectively, the results indicate small numbers of morphologically and functionally immature AMs prior to birth followed by a dramatic increase within the first 24 hr. This increase coincides with the large release from type II cells of surfactant, which not only may be involved in the functional maturation of developing AMs but also, if present in large quantities, may lead to decreased activity of these cells. There is also an age-related increase in chemotactic and microbicidal activity of AMs during maturation. Deficiencies in bone marrow pools, complement and cytokines (IFN-gamma, TNF-alpha) as well as IgG isotype switching are known to occur in the neonate which may contribute to increased susceptibility to infection and which are amenable to immune interventions.

Studies on gonococcus infection. VI. Electron microscopic study on in vitro phagocytosis of gonococci by human leukocytes.

The ingestion of Neisseria gonorrhoeae by isolated human leukocytes in vitro has been studied by electron microscopy. Use of the modified critical-point drying method allows clear visualization of progressive ingestion of both pilated and nonpilated gonococci by leukocytes. Incomplete ingestion of pilated gonococci with protrusion of tangled masses of pili from sites of phagocytosis are also found.

Current concepts of immune interventions in children with respiratory diseases.

In addition to their role in oxygen transport and ventilation, the lungs serve as an important defense function consisting of nonspecific and specific components. The nonspecific factors include aerodynamic filtration, mucociliary apparatus, bronchoalveolar fluid flow and lymphohematogenous drainage (anatomic systems) as well as phagocytosis and inflammation; the specific factors include B-cell immunity (IgG, A, M, D and E) and T-cell immunity (cell-mediated immunity). In the lungs, specialized lymphoid tissues in contact with epithelium (bronchus-associated lymphoid tissues; BALT) function in local antibody (secretory IgA) and cell-mediated immunity responses to foreign antigens. Based upon these considerations, a number of therapeutic interventions have been developed to enhance various components of lung defense. These include substances which enhance both nonspecific elements (leukocyte transfusion, plasma, nonspecific immunostimulants, e.g., immunoactive bacterial extracts) as well as specific elements (vaccines, intravenous gammaglobulin, plasma, interferons, cytokines). The need for further development and utilization of new immune interventions is underscored by the large number of infants and children who suffer from recurrent respiratory infections, who have either maturational immaturity (e.g. small for gestational age newborn), genetically determined (e.g. cystic fibrosis) or acquired defects (e.g. AIDS) of lung defense mechanisms. The emergence of antibiotic-resistant bacterial organisms, e.g. Streptococcus pneumoniae, poses an additional need for new immune interventions.

Studies on gonococcus infection. VII. In vitro killing of gonococci by human leukocytes.

The comparative killing of pilated and nonpilated forms of Neisseria gonorrhoeae by human peripheral blood leukocytes was studied in vitro. Some nonpilated gonococci (T2) were killed to a lesser extent than were pilated, T2 organisms, which were killed less readily than another nonpilated (T4*) form of gonococcus. Thus, the relative order of killing of gonococci by human peripheral blood leukocytes appears to be: T4 less than T2 less than T4*. These data suggest that pilation, though correlated with virulence of gonococci, has little influence on the survival or killing of these organisms by human leukocytes.

Studies on gonococcus infection. VIII. 125Iodine labeling of gonococci and studies on their in vitro interactions with eukaryotic cells.

Intact gonococci (GC) have been labeled with 125iodine by the lactoperoxidase plus hydrogen peroxide procedure. The specific activities of types 2, 4, and 4 GC have been determined and are found to show small differences as follows: T4 greater than T2 greater than T4. 125I-labeled GC have been studied for their associations with both leukocytes and tissue culture cells. 125I-labeled GCshow the following relative order of association with the leukocytes: T2 equals T4* greater than T4. This contrasts with the relative degree of interaction between the GC and tissue culture cells, which follows the relative order: T2 greater than T4 equals T4*. Trypsin pretreatment of GC markedly reduces the association of all three types (T2, T4 AND T4*) with leukocytes but does not alter the level of attachment of any of the gonococcal types with tissue culture cells.

Functional and ultrastructural changes in alveolar macrophages from rabbits colonized with Bordetella bronchiseptica.

Alveolar macrophages from rabbits colonized with Bordetella bronchiseptica in their respiratory tract exhibited significant decreases in cell adherence, phagocytic uptake, and bactericidal activity compared with macrophages from uncolonized animals. These dysfunctions were accompanied by ultrastructural changes, including a decrease in overall cell density, a vacuolation of the rough endoplasmic reticulum, and an increase in organelle-poor cell surface projections.

Proliferation of alveolar macrophages in hyperoxia.

The effect of oxygen on the proliferative response of alveolar macrophages (AMs) was investigated. Alveolar macrophages cultured in hyperoxic atmosphere (95% O2 + 5% CO2) for 18 hours showed increased incorporation of [3H]-thymidine and proliferation in contrast to those cultured in a control atmosphere (95% air + 5% CO2). The proliferating cell was shown to be a macrophage by morphology, esterase staining, and phagocytic ability. The results suggest an oxygen-induced proliferation of AMs that may play a critical role in AM influx into the alveoli particularly at times of hyperoxia, eg, the neonatal period.

Chemotactic and candidacidal responses of rabbit alveolar macrophages during postnatal development and the modulating roles of surfactant in these responses.

Chemotactic responses of alveolar macrophages from 1-, 7-, and 28-day-old rabbits to various concentrations of endotoxin-activated serum and n-formyl-methionyl-phenylalanine were tested utilizing both blind well and agarose plate assay systems. A dramatic increase in both the chemotactic response and responsiveness to various concentrations of chemoattractant was observed during postnatal maturation. The pattern of result was similar with both methods of assay. An age-related increase was also found to occur in the candidacidal activity of alveolar macrophages in contrast to their phagocytic uptake, which showed no age-related increases. Furthermore, the decreased function of macrophages from newborn animals correlated with a morphologically and biochemically less mature cell population which contained large amounts of phagocytosed surfactant-related material. Moreover, pretreatment of macrophages from 7- and 28-day-old animals with vesicles of surfactant-related material resulted in decreases in both chemotactic and candidacidal activity, with a paradoxical increase in their phagocytic activity. The resulting activities were similar to those of macrophages from 1-day-old animals treated with buffer alone. These data suggest that there is an age-related increase in the chemotactic and candidacidal activity of alveolar macrophages during maturation and that the decreased activity of macrophages from newborn animals is related in part to the large amount of surfactant-related material present at that time.

Chemotactic responses of various differentiational stages of neutrophils from human cord and adult blood.

Chemotactic response and responsiveness, that is, the ability to respond to various concentrations of the chemoattractant zymosan-activated serum was measured for cord and adult blood neutrophils. Chemotactic response of neutrophils from adult blood was two to seven times greater than that of neutrophils from cord blood. In addition, neutrophils from cord blood showed poor response to concentrations of 25% chemoattractant or less, compared to those from adults, which responded to concentrations of 10%. Further, it was found that approximately 30% of neutrophils in cord blood were the band form compared to only 9% in adult. Based upon this, a simple method was devised to assay mean migrating distance of various differentiational stages of neutrophils incorporating both distance and magnitude of their responses. The results of these assays showed that all differentiational stages of cord blood neutrophils have a mean migrating distance less than those of adults. In addition, the band form from both cord and adult blood had a mean migrating distance less than the polymorphonuclear form. Adherence studies revealed that all differentiational stages of neutrophils adhered in a similar manner, and no difference was detected between cells from cord and adult blood. Assays to test the ability of cord blood to produce chemotactic activity when activated revealed 50% less activity than that obtained from adult serum which further decreased with dilution of attractant. These data suggest that both the cellular and humoral systems involved in chemotactic responses are less in cord blood compared to adult blood.

Maturation of the rabbit alveolar macrophage during animal development. III. Phagocytic and bactericidal functions.

Phagocytic and bactericidal function of rabbit alveolar macrophages (AMs) lavaged from animals during the course of postnatal maturation was studied. Staphylococcus aureus and a temperature-sensitive mutant of Escherichia coli, which could not replicate at 37 degrees during the functional assays, were employed as test bacteria. Assays of the phagocytic capacity of AMs from rabbits of various age groups revealed no significant differences either in the percentage of AMs which took up bacteria (79-90%) or in the number of bacteria taken up per AM (Table 1). In contrast, bactericidal activity of AMs was found to increase with increasing animal age. No bactericidal activity was detected in AMs from newborn animals (Figs. 1 and 2), whereas AMs from 7-day-old animals exhibited at least a bacteristatic activity against S. aureus (Fig. 1) and AMs from 28-day-old rabbits showed marked bactericidal activity, essentially the same as that of AMs from adult rabbits. Adult AMs killed 75% of the S. aureus and 60% of the E. coli within 120 min (Figs. 1 and 2).

Chronic granulomatous disease in an adult.

We have described a 49-year-old man with chronic granulomatous disease. The diagnosis was established by a deficiency of NBT dye reduction by neutrophils, in addition to impairment in 14C-1-glucose utilization, 125I-iodination of zymosan, chemiluminescence, superoxide radical generation, and bactericidal activity toward S aureus. This adult patient exhibits many characteristics of chronic granulomatous disease of childhood but of less severity, which may explain his unusually long survival. It is thus important to consider the diagnosis of chronic granulomatous disease not only in children but also in adult patients having the characteristic pattern of recurrent infections.

Ideal target organism for quantitative bactericidal assays.

We have developed a target organism which permits quantitative bactericidal assays. The organism is an Escherichia coli mutant which cannot grow at the temperature of the assay (37 degrees C), but retains full colony-forming potential for subsequent quantitation at 25 degrees C. We show that quantitative data on the bactericidal capacity of polymorphonuclear leukocytes and alveolar macrophages can be obtained when this mutant is used as a target. The procedure used to generate the strain is described in detail and should be applicable to many bacterial species. Characterization of the properties of the mutant indicates that it has a strong potential for use in other in vivo and in vitro investigations of host responses to microbial invasion.