Comparable investigations of the chemotactic and chemokinetic responses of purified monocytes (MO) and polymorphonuclear leukocytes (PMN) to leukotriene B4 (LTB4) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) were made in this study. Using a sensitive and objective 51Cr-chemotactic assay, it was shown that both MO and PMN showed a bell-shaped response to LTB4 and FMLP, with a maximum response at 10(-8)M for both drugs. For PMN, the maximal response elicited by LTB4 was similar in magnitude to that produced by FMLP, whereas the MO chemotaxis induced by 10(-8)M FMLP was significantly higher than the response evoked by 10(-8)M LTB4. For both cell types, LTB4 at low concentrations (less than 10(-9)M) gave rise to higher chemotactic responses than FMLP. Chemokinesis was differentiated from chemotaxis, using a checkerboard system. At concentrations less than 10(-9)M the LTB4-evoked contribution of chemokinesis to the total migrational response was significantly higher than the chemokinetic contribution of FMLP. Preincubation with LTB4 produced only homologous chemotactic deactivation to subsequent LTB4 stimulation, whereas preincubation with FMLP resulted in diminished secondary response to both FMLP and LTB4. The degree of deactivation was dependent upon the dose of attractant used, with a LTB4 concentration of 10(-7)M leading to about 40% and 25% deactivation of PMN and MO, respectively. Preincubation with 10(-7)M FMLP led to about 50% and 32% suppression of the subsequent chemotactic response of PMN and MO, respectively.
Highly purified normal human tonsillar B lymphocytes were cultured with recombinant IL 2 (rIL 2). The responses were minimal when the cells were cultured with rIL 2 alone. Dose-dependent proliferation and differentiation into immunoglobulin-secreting cells were found when the cells were simultaneously activated with Staphylococcus aureus Cowan I (SAC). T cells were not found to proliferate in these cultures. B-cell responses were not augmented when 16% T cells were added to the B-cell population. In contrast, B-cell proliferation and Ig secretion were significantly enhanced by adding 0.5% T cells when cultured in the presence of pokeweed mitogen (PWM). Moreover, B-cell stimulation by rIL 2 was detected at very low cell densities. It is concluded that IL 2 directly affects the function of activated B cells.
Binding of labelled protein to EIgM kept with macrophage or monocyte cultures with 3H-leucine under serum-free conditions, shows that de novo synthesis of protein with affinity to EIgM takes place. We find that monoclonal anti-C3c and anti-C3g antibodies and polyclonal anti-C4 and anti-C5 antibodies bind to such erythrocytes. This demonstrates that C4b, C3b and iC3b are deposited on the EIgM. Additional evidence for complement synthesis is the increase in binding of anti-C4 antibodies to EIgM when the incubation time was increased from 48 to 96 hours. Stimulation of the mononuclear phagocyte cultures with ET was necessary to obtain significant amounts of erythrocyte-bound complement proteins. From these results we conclude that the functional classical pathway of complement is produced in vitro by the monocytes and macrophages.
Analysis of synovial fluid from patients with rheumatoid arthritis (RA) and a long history of RA, who were undergoing synovectomy and joint surgery, revealed that 71 ± 15% of the mononuclear cells in the synovial fluid were lymphocytes. 3 ± 2% lymphoid blast cells, and 26 ± 15% were monocytes/macrophages. The lymphocyte population consisted mainly of T cells, and 72 ± 15% of lymphocytes were OKT11‐positive. OKT8‐positive cells (48 ± 9%) dominated over OKT4‐positive (32 ± 8%) cells with a mean OKT4/OKT8 ratio of 0.69. On the basis of la‐and Tac‐markers, very few of the lymphocytes were activated. Few B cells (12 ± 7%) and no plasma cells were seen. The results are discussed in relation to previous reports.
An isotype‐specific ELISA for detection of rheumatoid factors (RFs) is described. The assay utilizes rabbit anti‐albumin IgG bound to albumin as solid phase reactant (test wells). RF‐activity was expressed as the difference in reaction between test wells and wells containing F(ab′) 2 ‐fragments of the rabbit IgG bound to albumin. The activity correlated well with the conventional Waaler agglutination test on sera that showed a positive Waaler titre, but several sera that were negative by the Waaler test were positive for RF of IgG isotype by the ELISA‐test. The method was applied on specimens from psoriatic patients, and RFs of IgG‐and IgA isotype were demonstrated in serum immune complexes from all the psoriatic patients examined. IgG‐ and IgA‐RF was also demonstrated in extracts from psoriatic scale, and RFs in scale were demonstrated independently of free rheumatoid factor activity in serum.
To explore whether the intravenous administration of naturally occurring high-titer human anti-lipopolysaccharide IgG (anti-LPS) is beneficial in the treatment of human septic shock, it is necessary to select blood donors according to anti-LPS concentrations. The present paper describes an enzyme-linked immuno-adsorbent assay (ELISA) for the screening of blood donors to obtain such antibodies. Human specific IgG antibodies directed against smooth LPS from eleven different bacterial strains and species were quantitated. The ELISA is simple and uses only commercially available reagents. By optimizing assay conditions, a low unspecific background activity (OD below 0.010) was obtained, together with a large specific reading interval. 2021 human plasma samples from volunteer blood donors at six different blood-banks in Denmark were screened by the ELISA. The median anti-LPS antibody concentration was 9 mg/l. Five per cent of the donors had anti-LPS concentrations above 40 mg/l (expected to be therapeutically useful). The age- and sex-related distributions indicated that the highest prevalence of high anti-LPS concentration was in males aged 40-49 years. It is concluded that specific anti-LPS gammaglobulin based on naturally occurring anti-LPS antibodies can be obtained in Denmark following the screening of blood-donors, using the assay developed.
In order to study the regulation of the human B cell response to pneumococcal polysaccharides (PPS), anti-PPS-secreting cells (SC) and Ig-SC were quantitated in unstimulated and EBV-stimulated cultures of blood mononuclear cells (MNC) established before and one and two weeks after vaccination. In unstimulated cultures established one week after vaccination, significant numbers of anti-PPS-SC were detected; however, they were observed only in cultures depleted of T cells (mean: 62/10(6)), suggesting the presence of T cells suppressing in vivo-activated PPS-specific B cells. In EBV-stimulated cultures depleted of T cells, low numbers of anti-PPS-SC were observed before the vaccination, and the numbers of these cells increased significantly two weeks after the immunization (from 28 to 48/10(6)). In contrast, the numbers of anti-PPS-SC and total Ig-SC decreased in EBV-stimulated cultures of unseparated MNC established after the vaccination, suggesting T cell-mediated suppression. The findings were not explained by changes in the numbers of blood T helper (Leu 3+) or T suppressor (Leu 2+) cells following the vaccination. Possibly the vaccination induces the appearance in the blood of activated suppressor cells responsible for the suppression of B cells in unstimulated and EBV-stimulated cultures.
Previous electron-microscopic studies of isolated islets of Langerhans exposed to the monokine interleukin-1 for 7 days have indicated that interleukin-1 is cytotoxic to all islet cells. To study the time-course and possible cellular specificity of interleukin-1 cytotoxicity to islets exposed to interleukin-1 for short time periods, isolated rat or human islets were incubated with or without 25 U/ml highly purified human interleukin-1 for 24 h. Samples of rat islets were taken after 5 min, 30 min, 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 h and samples of human islets after 5 min, 30 min and 24 h of incubation and examined by electron microscopy in a blinded fashion. Already after 30 min, accumulation of opaque intracytoplasmic bodies without apparent surrounding membranes, and autophagic vacuoles were seen in about 20% of the beta cells examined in rat islets exposed to interleukin-1. After 16 h of incubation with interleukin-1, more than 80% of rat beta cells showed signs of degeneration. Beta cell specific changes similar to those observed in rat islets exposed to IL-1 for 30 min were seen in human islets exposed to IL-1 for 24 h. The described changes were not observed in alpha cells in interleukin-1-treated rat or human islets, or in alpha and beta cells in control islets. Passing interleukin-1 over columns containing Sepharose-coupled anti-interleukin-1 antibody completely removed the beta cell cytotoxic action on rat islets.(ABSTRACT TRUNCATED AT 250 WORDS)
T4 cells recognize antigen together with HLA class II molecules in the membrane of antigen-presenting cells (APC). The magnitude of the induced T cell response is in part dependent upon the quantitative expression of HLA-class II molecules in the cell membrane of the APC. We report here that recombinant interferon gamma (IFN-gamma) may enhance the expression of HLA class II molecules on the surface membrane of blood-derived dendritic cells by 30-90%.
Serum IgG1, IgG2, IgG3 and IgG4 antibody levels directed against lipopolysaccharide (LPS) from Bacteroides gingivalis were measured in the sera from systemically healthy subjects with and without periodontitis. An enzyme-linked immunosorbent assay was used that included coating of microtiter plates with LPS, and subsequent incubation with patient sera followed by mouse monoclonal subclass-specific antibodies, biotinylated sheep anti-mouse IgG and alkaline phosphatase conjugated to streptavidin. Anti-LPS IgG antibodies were dominated by IgG2, and moderate amounts only of IgG1, IgG3 and IgG4 were found. The periodontitis patients had significantly higher anti-LPS IgG1, IgG2 and IgG3 levels when compared to the subjects with healthy periodontium (p less than 0.05, Mann-Whitney test).
The production of serum opsonins and other antibodies to serogroup B serotype 15 meningococci was examined in 7 patients with serogroup B serotype 15 meningococcal disease and 7 volunteers immunized with a vaccine containing outer membrane proteins from serogroup B serotype 2b and 15 meningococcal strains complexed with polysaccharides from serogroups A, C, Y and W-135 meningococci. Serum opsonic activity was measured by a flow cytometric phagocytosis technique, using unfixed serogroup B Neisseria meningitidis labelled with fluorescein isothiocyanate. Serum antibodies to outer membrane complexes prepared from the meningococcal test strain, B:15:P1.16, were measured by an enzyme linked immunosorbent assay. The mean number of bacteria per phagocyte increased from 9.7 to 17.3 (mean difference 7.6, p less than 0.001) when the meningococci were opsonized with convalescent sera compared to sera obtained during the acute illness, and from 8.0 to 15.4 (mean difference 7.4, p less than 0.001) when opsonized with sera from immunized versus pre-immunized volunteers. The patients had insignificant amounts of serum antibodies to the group B meningococcal test strain on admission to hospital. Two weeks later all had a marked increase in IgG, IgM and IgA serum antibodies. Vaccination caused a marked increase in serum IgG antibodies. Serum opsonic activity and IgG levels were still high 6 weeks after the acute illness/immunization. A correlation was observed between serum opsonic activity and IgG antibody levels (r = 0.883, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
Peritoneal macrophages (PM luminal diameter) from untreated C57B1 mice contain high levels of beta-galactosidase (beta-gal) and these PM luminal diameter are heterogeneous in their expression of this enzyme. Intraperitoneal (i.p.) injection of saline caused a transient depression in the level of enzyme activity in the PM luminal diameter whereas i.p. injection of Proprionibacterium acnes (P. acnes) gave rise to a marked decrease of beta-gal activity in these cells. This reduction in enzymatic activity persisted for as long as the PM luminal diameter were activated for cytotoxicity towards the L929 tumor cell line, up to 35 days after injection. beta-gal activity was present in the lavage fluid from day 2-21 after injection of P. acnes but none was detected in the lavage fluid after injection of saline. It is proposed that the enzymatic activity in the lavage fluid is derived from monocytes which migrate from the blood into the peritoneal cavity. There was an influx of granulocytes in the P. acnes group which persisted up to 35 days after injection. In contrast none were observed in the saline group after 2 days. PM luminal diameter harvested after 1-35 days were large, highly vacuolized and many contained bacteria; these PM luminal diameter had the typical morphology of activated cells. It is suggested that the processing of P. acnes by granulocytes may play a role in the activation of macrophages in the early inflammatory response, with concurrent loss in beta-gal activity. However, in the later stages, interferon-gamma and other induced lymphokines may be instrumental in causing a decrease in beta-gal activity.
Non-phagocytic adherent cells (NPAC) were found as contaminants in peritoneal macrophage cultures from mice kept in conventional animal houses. The cells were morphologically intermediates between macrophages and small lymphocytes. They showed a vigorous ameboid movement in vitro, and most of the cells detached during the first 24 h in culture. The cells were identified as B-lymphocytes by demonstrating surface immunoglobulin with fluorescence labelled goat-F(ab)2-anti-mouse IgG + IgM antibody, as non-phagocytes by latex beads phagocytosis test, and as non-T cells with an anti-Thy 1 monoclonal antibody. Macrophages were identified with M1/70 monoclonal antibody against the Mac-1 antigen. An increased number of NPAC was found in cultures harvested from the peritoneum of mice kept in conventional houses for one week or more, together with a rise in total peritoneal cell count. The keeping also resulted in increased functional activity of the macrophages, tested by morphology, lososomal enzyme activity and phagocytic functions. The rise in NPAC parallelled the activation of macrophages and may therefore serve as an indication of the functional state of the macrophage cultures.
Live or heat killed (30 min at 56 degrees C) Chlamydia psittaci elementary bodies (EB) were phagocytosed by mouse peritoneal macrophages. Inoculation with killed Chlamydia caused rises in three lysosomal enzyme activities tested, especially in acid phosphatase activity. In contrast, after infection with live Chlamydia, only a negligible increase was seen in acid phosphatase activity, and no change in the activities of beta-glucuronidase and cathepsin D was observed. It was concluded that regulation of lysosomal enzyme synthesis in macrophages may be linked to signals mediated by phago-lysosome fusions.
The recovery and purity of monocytes were assessed after adherence of human blood mononuclear cells to plastic surfaces, with subsequent detachment after exposure to EDTA, lidocaine, trypsin or low temperature (4 degrees C). No differences in purity were observed (81-90%) but the recovery was highest after treatment with lidocaine and trypsin. Pretreatment of the plastic surface with fetal calf serum increased the recovery to approximately 60%. The chemotactic response of the manipulated monocytes was decreased when trypsin had been used, compared with monocytes detached after exposure to low temperature, lidocaine or EDTA. In conclusion, for the measurement of chemotaxis the best results are obtained with monocytes after lidocaine detachment from a serum-pretreated plastic surface.
The effects of human leukocyte, porcine spleen and bovine liver dialysate fractions on Listeria resistance were measured by survival studies and by assessing the capacity of peritoneal macrophages to produce superoxide anion (0-2) and prostaglandins. Leukocyte (DLE) and other tissue dialysates were fractionated on a Sephadex G-10 column. Thereafter the significant activities were found in fraction III of DLE, fraction II of porcine spleen and fraction II + III of bovine liver dialysate. The treatment with active porcine spleen dialysate fraction increased the capacity of peritoneal macrophages to generate superoxide anion. On the other hand, this fraction significantly decreased the production of prostaglandin PGE2 and thromboxan B2. These results may indicate that all dialysates can be a source of a non-specifically-acting immunomodulatory preparation and that the infection-resistance-increasing substances seem to operate via the monocyte/macrophage activation.
A selective zymosan-induced release of lysozyme from freshly prepared human blood granulocytes and monocytes was inhibited by indomethacin, sulindac, piroxicam and ibuprofen. This effect was slightly more marked for granulocytes than for monocytes. Salicylic acid, acetylsalicylic acid and naproxen, even at high concentrations, did not inhibit the enzyme release from either cell type. Since all these nonsteroidal anti-inflammatory agents are cyclooxygenase inhibitors, these findings suggest that lysozyme release is independent of prostaglandin biosynthesis.
Nine strains, isolated from leukoplakias or normal mucosa of the oral cavity, and representing the species Candida albicans, C.tropicalis, and Torulopsis glabrata were tested for the capacity to degrade IgA1, IgA2, and S-IgA in liquid cultures. IgA fragments were characterized by SDS-PAGE of culture supernatants in combination with immunoblotting analysis using antibodies specific for heavy chain and light chain determinants. Strains of C.albicans and C.tropicalis were found to express stronger proteolytic activity than a strain of T.glabrata. The three types of IgA were all degraded, alpha-chains being the primary targets. Immunoblotting analysis indicated that divalent fragments corresponding to the deletion of one or both of the Fc alpha constant domains (F(abc)2 alpha or F(ab)2 alpha) were produced. Monovalent half-molecules corresponding to these fragments could also be detected, suggesting that the yeast strains were capable of cleaving inter-alpha-chain disulphide bridges. The possible consequences of yeast-induced degradation for the function of IgA antibodies are discussed.
Sixteen complement components were analysed at three different occasions during a normal pregnancy. Samples were also obtained at delivery and six weeks post partum. All factors but C1 IA and D increased during the pregnancy. Six weeks after the delivery, the levels of all components but C1s, C4 and C6 showed a beginning normalisation. In 15 per cent of the women, C1 IA was functionally inactive at delivery.
In order to obtain specific tools for studying the alterations of the immunochemical structure of Yersinia enterocolitica lipopolysaccharide in various conditions, we have produced monoclonal antibodies reacting with core and O-polysaccharide chains of Yersinia enterocolitica O:3 LPS. Immunizations were made with whole bacterial cells and outer membrane preparation, respectively. Monoclonal antibody 2B5 reacted in enzyme immunoassay with purified core-lipid A complex, and its binding was not inhibited by Polymyxin B, suggesting that the target determinant is in the outer core. 2B5 recognized 100% of all tested Y. enterocolitica O:3 strains (n = 152) and reacted to some extent also with many other gram-negative bacteria. In immunoblotting with 2B5, a band corresponding to core-lipid A complex was visualized both with Y. enterocolitica, Brucella abortus and Haemophilus influenzae. In immunofluorescence assay, the only positive reaction was seen with Y. enterocolitica. Monoclonal antibody A6 reacted in enzyme immunoassay with purified O-polysaccharide chains, recognized 100% of tested Y. enterocolitica O:3 strains, and showed no cross-reactions with other bacteria. A typical ladder pattern was not seen in the immunoblotting analysis with A6. This suggests that the O-chain of Y. enterocolitica O:3 may be different from those in other gram-negative bacteria. These two antibodies will make it possible to study the structural variations of Yersinia enterocolitica LPS more precisely than described before, because of their fine specificity against important immunogenic components of LPS. They will also be useful in serology measuring the immune response against the target determinants of these antibodies.
