Plasmodium chabaudi chabaudi: B-1 cell expansion correlates with semiresistance in BALB/cJ mice.

The largest obstacle impeding the development of an effective malaria vaccine is the incomplete understanding of how the immune response is regulated during infection. B-1a cells, a poorly understood subcategory of B lymphocytes, produce nonpathologic autoantibodies of low affinity which have been shown to have distinct immunoregulatory capabilities. What the exact activity of B-1a cells are during the course of malaria has yet to be determined. By use of flow cytometry, it was observed that B-1a cells significantly expand by day 3 postinfection in the spleen and peritoneum of Plasmodium chabaudi chabaudi semiresistant BALB/cJ mice, but not until day 8 postinfection in the spleen of P. chabaudi chabaudi fully susceptible BALB/cByJ mice. The activation of B-1a cells was also demonstrated by the measurement of natural autoantibody IgM production from the serum and cultured peritoneal B-1a cells. Infected semiresistant BALB/cJ mice generated higher levels of anti-ssDNA IgM antibodies than infected fully susceptible BALB/cByJ mice. The preliminary data presented here suggest a possible roll of B-1 cells in contributing to the successful survival of murine malarial infection.

Immunological characterization of antigens released by Trypanosoma cruzi-infected cells.

Chagas' disease, caused by Trypanosorna cruzi, is characterized by the appearance of pathological lesions in the heart and other tissues during the chronic phase. The mechanisms responsible for such damage are still unclear. In the vertebrate host, T. cruzi replicates intracellularly before transforming from amastigotes into trypomastigotes. The infected host cell then lyses, releasing the cytoplasmic contents and the parasites that shed membrane glycoproteins soon after release. The sum of all these components we have termed released antigen (Rag). We characterized antigens, released in vitro by fibroblasts infected with T. cruzi, obtained by concentrating conditioned serum-free culture media. The results demonstrate that Rag contains a complex protein mixture including stage-specific T. cruzi antigens (Ssp-1, -2, -4), glucose-regulated protein (Grp) 78h, and peptides recognized by the monoclonal antibody 2B10. These peptides exhibit neuraminidase activity and are expressed by intracellular and 10-20% of released trypomastigotes. Additionally, Rag is recognized by sera from T. cruzi-infected mice and human chagasic patients. Rag also stimulates in vitro production of interferon-gamma by splenocytes from resistant C57B1/6 and susceptible BALB/c infected mice and interleukin-4 by splenocytes from BALB/c infected mice. Altogether these results indicate that Rag is immunologically relevant and could contribute to pathogenesis of T. cruzi infection.

Interleukin-12 and tumor necrosis factor alpha mediate innate production of gamma interferon by group B Streptococcus-treated splenocytes of severe combined immunodeficiency mice.

The existence of interleukin-12-mediated innate immune responses to group B streptococci (GBS) was tested by examining T-lymphocyte-independent gamma interferon (IFN) production in cultured splenocytes from severe combined immunodeficiency mice. Splenocytes were cultured with killed or living GBS for 48 h, and then IFN was measured by enzyme-linked immunosorbent assay. Type III GBS as well as other extracellular bacterial agents of neonatal sepsis (staphylococci and enterococci) induced IFN production, which was enhanced by interleukin-2 and was inhibited by neutralizing antibodies to tumor necrosis factor alpha and to mouse interleukin-12. Interleukin-12 bioactivity was present in conditioned medium from GBS-treated adherent macrophages. Adherent peritoneal macrophages and bone marrow-derived natural killer cells from severe combined immunodeficiency mice cultured separately with GBS did not produce IFN, whereas cocultures did produce IFN. Functional macrophage activation was evident by nitric oxide production in GBS-treated splenocyte cultures. The results show that extracellular pathogens such as GBS, similarly to intracellular microbes, induce macrophage interleukin-12 and tumor necrosis factor alpha, which promote natural killer cell secretion of IFN, which then enhances innate phagocyte resistance mechanisms.

Tumor necrosis factor alpha acts as an autocrine second signal with gamma interferon to induce nitric oxide in group B streptococcus-treated macrophages.

Nitric oxide production by mouse macrophages treated with group B streptococci and gamma interferon was inhibited by cytochalasin B or by antibody neutralization of macrophage-derived tumor necrosis factor alpha. Phagocytosis-induced tumor necrosis factor alpha is responsible for group B streptococcus-induced nitric oxide production in interferon-treated macrophages.

Group B streptococcus-induced nitric oxide production in murine macrophages is CR3 (CD11b/CD18) dependent.

Nitric oxide (NO) is produced by murine macrophages in response to cytokines and/or gram-negative bacterial lipopolysaccharide. NO induction by gram-positive bacteria such as group B streptococci (GBS), the major etiologic agents of neonatal pneumonia and meningitis, has received little study. GBS as well as two other gram-positive bacterial species, Staphylococcus aureus and Staphylococcus epidermidis, were found to stimulate NO production in thioglycolate-elicited murine macrophages and in the mouse macrophage cell line J774A.1 in the presence of gamma interferon. Serotype Ia and III GBS were both stimulatory, as were asialo- and type antigen-deficient mutant strains of type III GBS. NO production was dose dependent, inhibitable by L-arginine analogs, and unaffected by polymyxin B. Since phagocytosis by murine and human phagocytes of GBS is dependent on complement receptor type 3 (CR3), the role of CR3 in the NO response to GBS was tested in the CR3-deficient myelomonocytic cell line WEHI-3. GBS did not induce NO, whereas S. aureus or lipopolysaccharide did induce NO in WEHI-3 cells. S. epidermidis, whose nonopsonic phagocytosis is also CR3 dependent, failed to induce NO in WEHI-3 cells. Monoclonal anti-CR3 (anti-CD11b or anti-CD18) in the presence of interferon also induced NO production in thioglycolate-elicited macrophages and in J774A.1 cells but not in WEHI-3 cells. This evidence suggests that ligated CR3 and gamma interferon act synergistically to induce NO production and that CR3 mediates the GBS-induced signal for NO production in interferon-treated macrophages.

Opsonin-independent phagocytosis of group B streptococci: role of complement receptor type three.

The role of complement receptor type 3 (CR3) in nonopsonic recognition of group B streptococci (GBS) by macrophages was investigated. Monoclonal anti-CR3 (anti-Mac-1) inhibited phagocytosis of GBS strains by as much as 50% in serum-free cultures of both mouse peritoneal macrophages and the macrophage cell line PU5-1.8. GBS uptake was unaffected by the presence of anti-C3 or salicylhydroxamate, an inhibitor of the covalent binding reaction of C3. Soluble antibodies to LFA-1 or to the common beta-chain (CD18) of the LFA-1/CR3/p150,95 family of cell adhesion molecules did not inhibit GBS uptake. Down-modulation of surface Mac-1 on macrophages following adherence to anti-Mac-1- or anti-CD18-coated surfaces also inhibited uptake of GBS. Further evidence for GBS interaction with CR3 was demonstrated by reduction of EC3bi rosette formation in macrophages adherent to GBS-coated plates. These studies suggest that GBS can interact with macrophage CR3, promoting phagocytosis in a C3-independent fashion. In the absence of specific immunity in neonates, this recognition mechanism may be a significant virulence determinant for GBS which poorly activate the alternate complement pathway.

Stimulation of complement component C3 synthesis in macrophagelike cell lines by group B streptococci.

Complement levels and complement activation are key determinants in streptococcus-induced inflammatory responses. Activation of macrophage functions, such as complement synthesis, by group B streptococci (GBS) was examined as a possible component of GBS-induced chronic inflammation. Using an enzyme-linked immunosorbent assay, secreted C3 from mouse macrophagelike cell lines (PU5-1.8 and J774A.1) was monitored after cultivation with GBS. Whole, heat-killed GBS (1 to 10 CFU per macrophage) of both type Ia and III strains induced 25 to 300% increases in secreted C3 in both cell lines after a 24-h cultivation. GBS-treated cell lines exhibited increases in secreted lysozyme (10%) and in cellular protein (25 to 50%). Inhibition of macrophage phagocytosis by cytochalasin B inhibited GBS stimulation of C3. Purified cell walls of GBS type III strain 603-79 (1 to 10 micrograms/ml) also enhanced C3 synthesis. Local enhancement of macrophage C3 production by ingested streptococci or by persistent cell wall antigens may serve to promote chronic inflammatory responses.

Complement component C3 secretion by mouse macrophage-like cell lines.

Secretion of complement component C3 by the mouse macrophage-like cell lines PU5-1.8, J774A.1, RAW264.7, and P388D1 was measured using an enzyme-linked immunosorbent assay for mouse C3. All cell lines secreted antigenically detectable C3 with the relative secreted C3/10(6) cells/24 h ranked as J774A.1 greater than P388D1 greater than or equal to PU5-1.8 much greater than RAW264.7. C3 secretion was enhanced two- to fourfold in cultures of all cell lines when treated with lipopolysaccharide, streptococcal cell walls, or lymphokine-containing supernatant fluids of mitogen-stimulated spleen cells. A differential induction of C3 synthesis and secretion was indicated since secreted lysozyme and total cellular protein were not elevated in a manner comparable to C3. The relative inducibility of cell lines for C3 secretion in either lipopolysaccharide- or cell wall-treated cells could be ranked as PU5-1.8 greater than P388D1 greater than J774A.1 greater than RAW264.7. C3 secretion was inhibited by cycloheximide or hydrocortisone. Mouse macrophage-like cell lines retain baseline and inducible C3 synthetic activities as do normal macrophages and can serve as homogeneous cultures in which to study regulation of complement biosynthesis.

Modulation of complement fixation and the phlogistic capacity of group A, B, and D streptococci by human lysozyme acting on their cell walls.

Streptococci and streptococcal cell wall fragments induce arthritis in rats, with the severity and duration depending on the capacity of the cells or cell fragments to resist degradation by tissue enzymes. Their phlogogenic effects are apparently related to their ability to activate the alternate complement pathway (ACP). The in vitro activation of the ACP by lysozyme-treated cells and cell walls of group A, B, and D streptococci suggests that both rat and human lysozyme can modulate this activity, i.e., increasing it, decreasing it, or doing both in that order. The effects of the lysozymes also correlated with the degree to which they can unmask the aminosugar-reducing groups detectable in a given amount of cell wall, which suggests that partial depolymerization of the cell wall is critical for ACP activation. The effects of mutanolysin and C phage lysin on ACP activation were found to be correlated with their action on streptococcal cell walls. Neuraminidase had relatively little effect on ACP activation by most streptococcal strains tested. We conclude that the participation of tissue enzymes, including but not necessarily limited to lysozyme, is an important determinant for the clinical arthritis induced by group A, B, or D streptococci. Experimental arthritis induced in rats with whole (or disrupted) streptococci may depend both on the capacities of the cell walls to activate the ACP and on the capacities of the host tissue enzymes to modulate this activation. Great severity and long durations of the disease were determined by the capacity of the enzymes to degrade cell wall antigens to a degree sufficient to ensure efficient activation of the ACP without completely degrading the material so that it no longer activates complement. In this model, the limited resistance of group B peptidoglycan to lysozyme was a critical pathogenic factor.

Toxicity of group B Streptococcus agalactiae in adult rats.

Several strains of group B Streptococcus agalactiae were found to be lethal for young adult rats. When bacteria were heat killed and then injected intraperitoneally into rats, rapid death (14 to 18 h) of the rats occurred, characterized by labored breathing, hemolyzed serum, hemoglobinuria, and subungual hemorrhages. Sections of tissues from these rats failed to reveal the cause of death. Rats injected with toxic or nontoxic strains of group B S. agalactiae had reduced numbers of circulating leukocytes and low serum C3 levels in comparison with those in control rats. The toxic strains of group B S. agalactiae induced dramatic decreases in platelet numbers, and in plasma fibrinogen levels as well, suggesting that the toxicity was due to disruption of the coagulation system. Rapid death in the absence of infection suggests that group B S. agalactiae may have a cell-associated toxin that induces these changes. Such a toxin may be a contributory factor in the high mortality rate associated with group B streptococcal infections of the human neonate.

Peritoneal macrophages of pathogen-free rats but not of conventional rats secrete elastolytic activity.

Elicited peritoneal macrophages from Sprague-Dawley rats conventionally bred and housed failed, as we have reported, to produce detectable elastolytic activity in culture. They did produce lysozyme and plasminogen activator. We now show that in contrast to these cells, macrophages from pathogen-free, barrier-sustained rats produced readily demonstrable elastolytic activity. Rats raised pathogen-free and subsequently housed conventionally for 2-4 wk appeared to lose the capacity to afford macrophages producing elastase. At the same time they acquired infections with several rat pathogens including Spironucleus muris, Kilham rat virus, sialodacryoadinitis virus, and mycoplasma pulmonis. The acquisition by the rats of one or more of these infections, conditions conducive to infection, or both factors may have suppressed their capacity to yield elastolytic activity.

Rat arthritis due to whole group B streptococci. Clinical and histopathologic features compared with groups A and D.

Heat-killed streptococci of Groups A, B, and D injected intraperitoneally into Sprague-Dawley rats induced arthritis. The histopathologic features of the arthritis were those of erosive synovitis. Early acute lesions were associated with deposits of streptococcal antigens. The serogroups and the physical state of the streptococci determined the incidence, the time of onset, the duration, and the severity of the disease, the severity being a blend of degree of inflammation, tendency to relapse, and occurrence of ankylosis. Whole Group A usually failed to induce arthritis. Group A disrupted with sonication regularly induced arthritis after a 24-hour latent period. The disease lasted over 60 days and caused ankylosis. Whole Group B regularly induced arthritis but only after a latent period of 6-8 days. The disease lasted over 40 days and caused ankylosed joints. With sonicated Group B a similar disease was induced, except that, as with sonicated Group A, the latent period was 24 hours. Whole Group D induced disease after a latent period of 48 hours. The arthritis lasted only 2 weeks and was transient. In contrast to its effects on Group A and B cocci, sonication of Group D abrogated its capacity to induce arthritis. It is postulated that for whole streptococci, in contrast to sonicated streptococci, arthritogenicity depends on the sensitivity of the cocci to initial processing in vivo. Processing may be partial digestion by enzymes of phagocytes. Cocci such as those of Group A that are insensitive to processing, injected whole, tend not to cause arthritis, but when they do cause disease, it is chronic. A coccus, such as one of Group D, that is very sensitive to processing produces a transient arthritis after a short latent period, while a coccus of intermediate sensitivity, such as one of Group B, induces disease only after a substantial latent period, and the disease is severe and chronic. The nature of processing remains to be determined.

Selective modification of rat peritoneal macrophage lysosomal hydrolases by inflammatory stimuli.

Lysosomal acid hydrolases were surveyed in elicited and non-elicited rat peritoneal macrophages to determine the types of enzymes present and optimal assay conditions. Adherent peritoneal cells (primarily macrophages) were cultured 24 hours prior to use. Intracellular distribution of enzymes was determined by differential centrifugation of whole cell homogenates into nuclear, cytoplasmic, and lysosomal fractions. The acid glycosidase, acid phosphatase, acid protease, and lysozyme were largely sedimentable in the lysosomal fraction. Much enzyme activity was latent, being activated by addition of Triton X-100. Chymotrypsin-like protease activity in cell fractions was apparently due to low level mast cell contamination. Elicited macrophages had elevated total cell protein as compared to non-elicited cells, but changes in intracellular enzyme levels were selective depending on the enzyme and the stimulus used to elicit macrophages. Thioglycollate-elicited cells showed elevations of most acid hydrolases compared to non-elicited cells, whereas enzyme levels in zymosan-elicited cells were similar to those in non-elicited cells. All elicited cells showed marked decreases in total cellular alpha-D-mannosidase and alpha-L-fucosidase compared to non-elicited cells. Intracellular lysozyme levels also varied between different rat strains. Cultured macrophages exhibited increasing intracellular levels and extracellular secretion of acid hydrolases, especially extracellular lysozyme (10-25 mug/10(6) cells/day), over 72 hours. No significant intra- or extracellular elastinolytic activity was detected.

Bactericidal activity in fractionated granule contents from human polymorphonuclear leukocytes: studies with leukocytes from normal individuals.

Sephadex G-100 chromatographic fractions of granule extracts from normal human polymorphonuclear leukocytes, exhibiting differences from fractions previously obtained from leukemic polymorphonuclear leukocytes, possessed cationic proteins with distinct bactericidal activity against cell wall mutants of Salmonella typhimurium LT2.

The use of Reuber hepatoma cells for the study of a lipopolysaccharide-induced macrophage factor: glucocorticoid-antagonizing factor.

Endotoxin-stimulated glucocorticoid-antagonizing factor (GAF) was assayed by its specific inhibition of hydrocortisone-induced synthesis of phosphoenolpyruvate carboxykinase. Defined induction of phosphoenolpyruvate carboxykinase synthesis in hydrocortisone-treated rat hepatoma cells permitted reliable quantitation of GAF and analysis of the mechanism of cortisol antagonism. GAF was present maximally in serum 2 hours after endotoxin challenge in mice; however, GAF production could be suppressed by pretreating mice with indomethacin or cortisol. Endotoxin-tolerant mice were also nonresponsive to endotoxin-stimulated GAF production. Gel filtration on Sephadex G-200 resolved four regions of glucocorticoid-antagonizing activity in serum from endotoxin-poisoned mice, two of which were not present in normal serum. Cortisol antagonism by GAF resembled that of insulin; however, insulin differed from GAF in its ability to antagonize dibutyryl cyclic AMP. Unlike insulin, endotoxin-induced serum glucocorticoid-antagonizing activity was heat-labile at 70 degrees C. GAF antagonism of hydrocortisone was partially reversible but did not act in a competitive manner. Production of hepatoma growth inhibitory activity and glucocorticoid-antagonizing activity in serum were closely associated, indicating a common methanism for their generation.

Elicitation of endotoxemic effects in C3H/HeJ mice with glucocorticoid antagonizing factor and partial characterization of the factor.

C3H/HeJ mice were used to study the origin and nature of endotoxin-induced glucocorticoid antagonizing factor (GAF). In conventional mice GAF is believed to be responsible for a variety of effects that occur as a result of an injection of endotoxin, including the inhibition of hormonal induction of hepatic phosphoenolpyruvate carboxykinase and of glyconeogenesis. Responses in such animals are seen whether the endotoxin is extracted with phenol-water or with trichloroacetic acid. C3H/HeJ mice do not respond (or produce GAF?) after an intravenous injection of phenol-water lipopolysaccharide, but they react normally (produce GAF?) when given a trichloroacetic acid preparation. They also behave the same as conventional animals when injected with serum from poisoned normal mice, especially when the reticuloendothelial system of the donors has been activated by prior injections of Zymosan or heat-killed tubercle bacilli. The C3H/HeJ mice have been used, therefore, as assay animals to establish that peak levels of GAF appear in donor serum about 2 h after an injection of lipopolysaccharide, and it is produced intraperitoneally in C3H/HeJ mice given a mixture of endotoxin and peritoneal exudate cells derived from responder mice. GAF elutes from Sephadex G-200 along with markers of known molecular weight in the region of 100,000 to 200,000. It is inactivated by trypsin and by heating at 75 degrees C for 1 h.