Effect of Capsular Polysaccharide of Klebsiella pneumoniae on Host Resistance to Bacterial Infections

In normal mice, the total count of peritoneal leukocytes was markedly decreased after intraperitoneal (i.p.) injection of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) depending on the dosage injected. This decrease was mainly due to the depletion of macrophages, and a decrease in the number of lymphocytes occurred to a lesser extent. CPS-K in relatively smaller doses mobilized polymorphonuclear neutrophilic leukocytes (PMN) into the peritoneal fluid but it decreased them transiently in larger doses. In mice infected i.p. with a virulent strain of Salmonella enteritidis, there was an abundant emigration of PMN into the peritoneal fluid. When 200 μg of CPS-K was injected i.p. immediately before bacterial challenge, emigration of PMN was markedly delayed for 48 hr after infection. Associated with this suppressed emigration of PMN, the numbers of macrophages and lymphocytes in the peritoneal fluid were significantly less in mice treated with CPS-K than those in untreated control mice for 48 hr after infection. The numbers of both cell-associated and extracellular bacteria in the peritoneal fluid were markedly greater in mice treated with CPS-K than those in untreated control mice. In both in vivo and in vitro experiments, ingestion of bacteria by macrophages and PMN was not blocked by CPS-K or neutral CPS-K, the active substance responsible for the infection-promoting effect of CPS-K. It appeared that CPS-K somehow impaired the intraphagocytic bactericidal activity.     

Increased susceptibility to Escherichia coli infection in mice pretreated with Corynebacterium parvum

The contribution of activated macrophages to protection against Escherichia coli was studied in mice treated intravenously with Corynebacterium parvum 7 days before infection. C. parvum-treated mice showed increased phagocytic activity and enhanced resistance to Listeria infection. In contrast, these mice showed increased susceptibility to a subsequent challenge with E. coli that correlated closely with a reduction in the LD50 of lipopolysaccharide (LPS) in these mice. The peritoneal macrophages obtained from C. parvum-treated mice had a strong ability to phagocytize and kill E. coli in in vitro experiments. A rapid decline in the number of bacteria in the liver of C. parvum-treated mice was observed in the early period of infection. However, the number of bacteria in liver and spleen increased progressively to a lethal dose from 6 hr after infection. At this time, a significant increase in beta-glucuronidase, a lysosomal acid hydrolase, was found in the serum of these mice. In vitro experiments revealed that the peritoneal macrophages from C. parvum-treated mice were highly susceptible to the cytotoxic effect of LPS after 6 hr of incubation with LPS. It is suggested that the hypersensitivity of activated macrophages to the cytotoxic effect of endotoxin derived from E. coli may be partly responsible for the increased susceptibility of C. parvum-treated mice to E. coli infection.     

Effect of capsular polysaccharide of Klebsiella pneumoniae on host resistance to bacterial infections. I. Induction of increased susceptibility to infections in mice.

When Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, was injected intraperitoneally (i.p.) immediately before i.p. bacterial challenge, the survival time of mice infected with Salmonella enteritidis NUB 1 (virulent strain) was shortened and the mortality rate for mice infected with S. enteritidis NUB 31 (avirulent strain) was enhanced. The promotion of infection with S. enteritidis NUB 1 by CPS-K depended upon its dose, the effect of CPS-K being demonstrable up to as little as 0.2 mug per mouse. In the case of S. enteritidis NUB 31, the effect of CPS-K was detectable only when more than 20 mug per mouse was injected. As a result of enumeration of bacterial populations in the peritoneal washing, blood, liver and spleen, it was revealed that CPS-K promoted in vivo growth of S. enteritidis NUB 1 and NUB 31. In addition, CPS-K enhanced the mortality rate in mice infected with Streptococcus pyogenes or Streptococcus pneumoniae. The peak CPS-K effect on infection with S. enteritidis NUB 1 was seen when given immediately before bacterial challenge. The active substance responsible for the infection-promoting effect of CPS-K was neutral CPS-K, which is distinct from the O antigen and from acidic CPS-K (the type-specific capsular antigen). Preparations of neutral CPS-K isolated from the other three strains of K. pneumoniae exhibited a marked infection-promoting effect comparable with that of preparations from the Kasuya strain. Neutral CPS-K, with identical antigenicity to that from the Kasuya strain, has already been found to exert a strong adjuvant effect on antibody responses to various antigens in mice. No parallelism exists between infection-promoting activity and adjuvant activity of neutral CPS-K.     

Novel immunomodulators with pronounced in vivo effects caused by stimulation of cytokine release

Beta-1,3-D-polyglucose derivatives protect mice against otherwise lethal bacterial infections. This protective effect has been considered to be mediated through mononuclear phagocytes. By using radioactive labelling, we localized the beta-1,3-D-polyglucose derivatized microbeads (GDM) during the period following injection. The GDM was recovered mainly in the milky spots of the omentum. In animals treated with GDM, the total white cell number was significantly increased in peritoneal fluid of mice before and after challenge with E. coli. Bacterial counts in peritoneal fluid of GDM treated animals declined to zero after 24 h. In untreated animals there was a slight increase in bacterial counts until the animals died after about 12 h. Mouse peritoneal macrophages stimulated with GDM released significant amounts of IL-1 and PGE2. There was no significant release of TNF. Levels of IL-1 and PGE2 in peritoneal fluid increased significantly during the first 48 h after treatment with GDM. There was no increase of levels of TNF. After challenge with E. coli, the levels of IL-1, TNF, and PGE2 were significantly lower compared with control animals. In untreated animals the levels of IL-1 and TNF remained elevated until the animals died after about 12 h. These studies demonstrate that the raised levels of arachidonic acid metabolites after pretreatment with GDM or AG seems to inhibit the otherwise lethal elevation of IL-1 and TNF in body fluids which is seen in untreated animals.     

Effect of capsular polysaccharide of Klebsiella pneumoniae on the differentiation and functional capacity of macrophages cultured in vitro.

The effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) on the differentiation and functional capacity of macrophages cultured in vitro from various lymphoid tissues was investigated. In cultures of peritoneal cells, the number of macrophages did not change throughout incubation periods of from 1 hr to 3 days, and the addition of CPS-K had no affect. It appears therefore that CPS-K does not exhibit cytotoxic effects on macrophages. In cultures of spleen cells, only a small number of macrophages appeared within 1 hr, but the number of macrophages increased during further incubation. The addition of CPS-K to cultures of spleen cells at the start of incubation suppressed markedly the increase in the numbers of macrophage. This finding indicates that CPS-K blocks the process of the generation of macrophages, probably from their precursor cells in cultures of spleen cells. Only a small number of macrophages appeared in cultures of thymocytes or lymph node cells either with or without CPS-K. The phagocytic capacity of either peritoneal macrophages or macrophages generated in cultures of spleen cells was activated during incubation in vitro. Macrophages cultured in the presence of CPS-K for 24 hr or longer appeared to have an enhanced phagocytic activity, although the enhancement of their phagocytic activity by the addition of CPS-K was less marked in cultures of spleen cells than in those of peritoneal macrophages. Morphologically, macrophages in both cultures of peritoneal cells and spleen cells incubated in the presence of CPS-K for 4 days possessed much longer cytoplasmic processes than those incubated in the absence of CPS-K. From the present study, it appears that CPS-K exhibits dual effects on macrophage precursor cells and macrophages, a blocking effect on the differentiation from the former to the latter and an enhancing effect on the functional capacity of the latter.     

Nitric oxide production and iNOS mRNA expression in mice induced by repeated stimulation with live Fusobacterium nucleatum

There have been few studies on the detection of direct nitric oxide (NO) production and interferon-gamma (IFN-gamma) in vivo without using animal cell culture. We questioned whether NO and IFN-gamma could be produced at the site of infection. The peritoneal cavity of mice was used as the local infection model. NO and IFN-gamma in abdominal washings from these mice were measured directly at various times after injection of Fusobacterium nucleatum, a gram-negative rod periodontal pathogen. The mice were divided into three groups: those treated with live bacteria (LB), those treated with heat-killed bacteria (HKB) and those untreated: normal (N). These mice were compared on the basis of cell filtration, NO and IFN-gamma production by injection of live bacteria (LFn) or heat-killed bacteria (HKFn). In the LB group, the total cell number increased corresponding to an increase in neutrophils after injection of both LFn and HKFn. A low level of NO was constantly produced in abdominal washings, but a significant amount of NO was synthesized in the LB group only 12 hr to 24 hr after injection of LFn. At the same time iNOS enzyme activity and iNOS mRNA expression were detected. IFN-gamma, which may contribute to enhance NO production, was also secreted at a high level from peritoneal exudate cells (PEC) at 12 hr and 24 hr in the LB group by stimulation of LFn. At 12 hr and 24 hr, iNOS positive cells in the LB group by infection of LFn were identified and shown to contain mostly macrophages. These findings indicate that live bacteria play important roles in NO production by macrophages. It is suggested that NO may contribute to the inflammatory response during F. nucleatum infection in periodontitis.     

Protection against gram-negative bacteremia in neutropenic mice with recombinant granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates production of neutrophils in bone marrow and may decrease the incidence of infection during neutropenia. We evaluated the protective role of recombinant GM-CSF against Pseudomonas aeruginosa challenge in neutropenic mice. CD-1 mice treated with cyclophosphamide on days 1 and 2 of the experiment were given GM-CSF (1, 2, or 4 micrograms/day) starting at day 4 of the experiment according to the following protocol: 1) 1 microgram of GM-CSF 2 hr and 24 hr after challenge; 2) 1 microgram 24 hr before challenge, 2 hr and 24 hr after challenge; 3) 2 micrograms injected 24 hr before and 2 hr after challenge; 4) 2 micrograms given 24 hr before and 2 micrograms given 2 hr and 24 hr after challenge; 5) 4 micrograms administered 2 hr and 24 hr after challenge; and 6) saline and bovine albumin controls. The number of blood neutrophils by days 4 and 5 was similar for GM-CSF-treated and untreated animals. Survival was significantly greater in animals given 2 micrograms of GM-CSF at 24 hr before and at 2 hr and 24 hr after challenge with Pseudomonas. Neutrophils and splenic macrophages obtained from GM-CSF-treated mice (2 micrograms/animal) produced significantly greater amounts of O2- (204 +/- 36 nmoles/10(5) cells) than controls (21 +/- 10 nmoles/10(5) cells). Additionally, neutrophils and macrophages from GM-CSF-treated mice killed significantly more bacteria (P. aeruginosa) in vitro and had a greater number of C3b and Fc receptors (78 +/- 12% and 89 +/- 8%) than did cells obtained from control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions Between Macrophages of Guinea Pigs and Salmonellae I. Fate of Salmonella typhimurium Within Macrophages of Normal Guinea Pigs

A suspended cell culture procedure was described for the cultivation of guinea pig macrophages infected with Salmonella typhimurium. The fate of the intracellular bacteria was assessed by quantitative recovery of viable bacteria with 0.5% solution of sodium desoxycholate. Two strains of S. typhimurium with different degrees of virulence for mice were compared. There was an initial destruction of intracellular bacteria of both strains; however, the extent of this destruction differed. Approximately 1% of the avirulent bacteria initially phagocytized survived at the end of 4 hr, whereas approximately 8% of the virulent bacteria survived at the end of 3 hr. After this initial killing, the intracellular bacteria began to multiply at a logarithmic rate between 3 and 21 hr after phagocytosis, and then a stationary phase was attained. The rate of this multiplication was comparable for both strains.     

Electron microscopy of the in vivo internalization of virulent Chlamydia psittaci 6BC strain.

The internalization of virulent Chlamydia psittaci 6BC particles by wandering mononuclear phagocytes in the peritoneal cavity of intraperitoneally inoculated mice occurred asynchronously, i.e., fragile reticulate bodies (RB) appeared to be more readily phagocytized than the rigid elementary bodies (EB). Early damage of mononuclear phagocytes occurred after internalization of chlamydiae. This was followed by a decreased uptake of particles, and may explain the relatively long persistence (up to 6 h after inoculation) of free, extracellular, "swollen", and RB-like particles. Internalized particles within phagolysosomes showed varying degrees of disintegration. The subsequent influx of polymorphonuclear phagocytes and monocytes into the inflammed peritoneal cavity may explain the rapid disappearance of chlamydiae and their antigens from the peritoneal fluid. The alteration in ultrastructure of peritoneal cells and chlamydial parasites during the inflammatory process are discussed.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. I. Enhanced production of interferon and appearance of cytotoxin stimulated by capsular polysaccharide of Klebsiella pneumoniae or bacterial lipopolysaccharide.

Interferon production stimulated by the active substance (neutral fraction) of the capsular polysaccharide of Klebsiella pneumoniae (neutral CPS-K) in BCG-infected mice was compared with that by bacterial lipopolysaccharide (LPS). Prior infection with BCG increased the responsiveness of mice to the lethal effect of neutral CPS-K as well as to that of LPS. Associated with this, BCG-infected mice showed a markedly enhanced ability to produce interferon after stimulation not only by LPS but also by neutral CPS-K. In addition, a cytotoxic factor (cytotoxin) was found to be released in the serum of BCG-infected mice after injection of these inducers. The kinetics of production of interferon and cytotoxin stimulated by neutral CPS-K were very similar to those stimulated by LPS. The time pattern of cytotoxin production was not in parallel with that of interferon production. Interferon reached a peak 2 hr and cytotoxin 3 hr after injection with these inducers. Interferon and cytotoxin produced by neutral CPS-K showed essentially the same stabilities to heating at 56 C and to treatment at pH 2 respectively as those produced by LPS. Interferon was inactivated by heating at 56 C more rapidly than cytotoxin. Cytotoxin was inactivated by treatment at pH 2 for 24 hr, whereas interferon activity was well preserved after this treatment. These results suggest that both activities are the result of different substances.     

Differences in initial rate of intracellular killing of Salmonella typhimurium by granulocytes of Salmonella-susceptible C57BL/10 mice and Salmonella-resistant CBA mice

The contribution of granulocytes to differences in the innate susceptibility of mouse strains to infection by Salmonella typhimurium was assessed on the basis of the size and composition of the inflammatory exudate after i.p. injection of bacteria and the intracellular killing of the bacteria by exudate peritoneal cells and blood granulocytes of resistant CBA and susceptible C57BL/10 mice. The increase in the numbers of both peritoneal granulocytes and macrophages 24 hr after i.p. injection of various numbers of live S. typhimurium was two to four times higher in C57BL/10 mice (p less than 0.05) than in CBA mice. However, despite the larger number of phagocytes in the inflammatory exudate, the numbers of viable S. typhimurium in the peritoneal cavity 24 hr after injection was higher (p less than 0.01) in C57BL/10 mice than in CBA mice. Because the proportion of noningested bacteria was similar in the two mouse strains (less than 30%), these findings indicate a difference in the rate of intracellular killing of the bacteria by exudate peritoneal cells (greater than 75% granulocytes) of the two mouse strains. Subsequent determination of the initial rate of intracellular killing (Kk) of S. typhimurium revealed that after phagocytosis of the bacteria in vivo, exudate peritoneal granulocytes (harvested 24 hr after i.p. injection of 10(3) live S. typhimurium) of CBA mice killed S. typhimurium twice as efficiently (Kk = 0.014 min-1; p less than 0.01) as exudate granulocytes of C57BL/10 mice (Kk = 0.008 min-1) did. Similarly, the initial rate of intracellular killing of the ingested S. typhimurium by blood granulocytes of CBA mice (Kk = 0.017 min-1) was two times higher (p less than 0.01) than that of C57BL/10 mice (Kk = 0.007 min-1). These findings may be specific for S. typhimurium, because L. monocytogenes were killed with equal efficiency by exudate granulocytes and blood granulocytes of these mouse strains (p greater than 0.20). The results of the present study are relevant with respect to the innate resistance of mice to S. typhimurium, particularly during the initial phase of infection when the inflammatory exudate contains predominantly granulocytes.     

Effect of Capsular Polysaccharide of Klebsiella pneumoniae on Host Resistance to Bacterial Infections

When Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, was injected intraperitoneally (i.p.) immediately before i.p. bacterial challenge, the survival time of mice infected with Salmonella enteritidis NUB 1 (virulent strain) was shortened and the mortality rate for mice infected with S. enteritidis NUB 31 (avirulent strain) was enhanced. The promotion of infection with S. enteritidis NUB 1 by CPS-K depended upon its dose, the effect of CPS-K being demonstrable up to as little as 0.2 μg per mouse. In the case of S. enteritidis NUB 31, the effect of CPS-K was detectable only when more than 20 μg per mouse was injected. As a result of enumeration of bacterial populations in the peritoneal washing, blood, liver and spleen, it was revealed that CPS-K promoted in vivo growth of S. enteritidis NUB 1 and NUB 31. In addition, CPS-K enhanced the mortality rate in mice infected with Streptococcus pyogenes or Streptococcus pneumoniae. The peak CPS-K effect on infection with S. enteritidis NUB 1 was seen when given immediately before bacterial challenge. The active substance responsible for the infection-promoting effect of CPS-K was neutral CPS-K, which is distinct from the O antigen and from acidic CPS-K (the type-specific capsular antigen). Preparations of neutral CPS-K isolated from the other three strains of K. pneumoniae exhibited a marked infection-promoting effect comparable with that of preparations from the Kasuya strain. Neutral CPS-K, with identical antigenicity to that from the Kasuya strain, has already been found to exert a strong adjuvant effect on antibody responses to various antigens in mice. No parallelism exists between infection-promoting activity and adjuvant activity of neutral CPS-K.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

Study was made to clarify the experimental conditions for the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to exhibit maximum adjuvant effect on antibody production to bovine serum albumin (BSA) in mice. To obtain the maximum primary antibody response and also the strongest priming for a secondary response to BSA, 1000 μg of CPS-K had to be injected intramuscularly into the same or adjacent site of BSA injection within the period of 1 hr before to 6 hr after the BSA injection. The optimum amount of BSA giving the maximum antibody response and also the strongest priming under these experimental conditions was 15 mg. In mice thus primed, an extremely high secondary response was induced by injecting 0.5 mg of BSA 30 days after the initial injection. The minimum amount of CPS-K, to exhibit a strong adjuvant action, was 100 μg, which was equal to the minimum amount to induce immunologic paralysis to a homologous antigen. Extremely large amounts, such as 100 to 300 mg per mouse of BSA, were also strongly immunogenic when injected together with paralyzing doses of CPS-K. In vitro admixture of BSA and CPS-K before injection did not strengthen adjuvant action of CPS-K. Alum-precipitated BSA mixed with CPS-K was not more immunogenic than native BSA mixed with CPS-K. Addition of Freund's complete adjuvant to an injection of BSA and CPS-K mixture did not enhance the adjuvant effect of CPS-K.     

Protective effect of acidic mannan fraction of bakers' yeast on experimental candidiasis in mice

An acidic fraction of bakers' yeast mannan, WAM025, showed a significant protective effect against Candida albicans infection in mice, but a neutral fraction of the same bakers' yeast mannan, WNM, did not exhibit this effect. Moreover, pretreatment with WAM025 resulted in a marked reduction of proliferation of C. albicans cells in the organs of the infected mice. We investigated the stimulative effect of these mannan fractions on the function of mouse peritoneal phagocytes, and found that mice administered WAM025 showed a greater increase in the number of peritoneal exudate cells, macrophages and polymorphonuclear leucocytes (PMN), than the mice treated with WNM, especially in the proportion of PMN. Peritoneal phagocytes, PMN and macrophages obtained from WAM025-treated mice showed marked candidacidal activity. Of the phagocytes, PMN were responsible for the larger part of the candidacidal activity. The myeloperoxidase activities of PMN and macrophages in WAM025-treated PEC were greater than in untreated macrophages. The myeloperoxidase activity of WAM025-treated PMN was significantly greater than that of WAM025-treated macrophages. This activity paralleled the active oxygen-releasing activity of the phagocytes. On the other hand, the phagocytic activity of phagocytes from mice administered WNM or WAM025 for C. albicans cells was identical to that of untreated phagocytes. WAM025 seems to cause enhance elimination of the pathogen from mice, by increasing the number and candidacidal activity of phagocytic cells.     

Evidence for granulocyte-mediated macrophage activation after C. parvum immunization

It has been previously demonstrated that at the peak of the peritoneal response to Corynebacterium parvum (Day 4), cytolytic macrophages can be characterized by the presence of intracellular bacteria. In the present study, the role of neutrophils in the activation of peritoneal macrophages by C. parvum was investigated. Inflammatory neutrophils isolated 5 hr after ip administration of C. parvum were transferred to normal, syngeneic mice and the peritoneal macrophages of recipients harvested 4 days later were tested for cytoxicity against HeLa cells. Neutrophils isolated from mice 5 hr after C. parvum immunization were effective in inducing cytolytic macrophages. Less than 100-fold as much bacteria was needed to induce comparable levels of cytotoxic activity when introduced inside granulocytes. Neutrophils obtained from mice 48 hr after C. parvum injection or mononuclear cells were not good macrophage activators. Viable neutrophils were not required as freeze-thawed cells were able to activate macrophages in recipient mice. The intracellular distribution of C. parvum changed dramatically with time. Initially almost all bacteria were found within neutrophils. By 24 hr, many macrophages contained either bacteria or granulocytes which had ingested C. parvum. Pyridine extracts of C. parvum, which do not activate peritoneal macrophages when injected directly into mice, did not induce neutrophils capable of activating macrophages. The residue of pyridine-extracted C. parvum did induce neutrophils that could activate macrophages when transferred. The results suggest that processing of the bacteria by inflammatory granulocytes may be an obligatory step in macrophage activation by this agent. The peak response occurred earlier than T-cell immunity is usually observed and it is suggested that direct activation of macrophages via ingestion of neutrophils may represent the earliest stage of macrophage activation by C. parvum.     

Suppressive effect of streptomycin on the phagocytic activity of mouse peritoneal macrophages for Histoplasma capsulatum

Streptomycin inhibited the phagocytic activity of mouse peritoneal macrophages forHistoplasma capsulatum. The inhibitory effect was demonstrable following both in vitro and in vivo administration of drug. The observations from examination by direct smear were confirmed by culturing for viable phagocytized organisms. A simple and reproducible technique for the counting of viable phagocytized organisms was developed. Forty-eight hours in vitro treatment of macrophage cultures with 10 to 200 µg/ml of streptomycin produced a graded inhibition of phagocytic activity, minimal at 10 µg/ml and maximal at 200 µg/ml of streptomycin. The parenteral administration of streptomycin significantly reduced phagocytic activity of mouse peritoneal macrophages forH. capsulatum. Mice were treated daily with the subcutaneous injections of 5, 2.5 or 1 mg streptomycin or saline. At 7, 14, 21 and 28 days post-treatment phagocytic activity of macrophages obtained from these mice was tested. There was a progressive, dose-dependent decrease in the phagocytic activity of macrophages derived from streptomycin-treated mice.     

Induction of interleukin 1 by Legionella pneumophila in murine peritoneal macrophage cultures

Legionella pneumophila-induced production of both membrane-associated and secreted interleukin 1 (mIL-1 and sIL-1, respectively) was examined utilizing peritoneal macrophages from BALB/c mice. The Legionella preparations for these studies included viable bacteria and formalin-killed whole cell preparations. Both of the preparations induced mIL-1 and sIL-1 in a dose-dependent fashion. However, the viable bacteria required about 1 log lower concentrations than the formalin-killed bacteria to induce the same level of IL-1 activity measured in the thymocyte proliferation assay. Kinetic studies showed that mIL-1 and sIL-1 were detectable within 4 hr after addition of either of the L. pneumophila preparations to the peritoneal macrophage cultures, with peak levels achieved within 24 hr. These results indicate that L. pneumophila is a potent inducer of both mIL-1 and sIL-1 in normal mouse peritoneal macrophage cultures.     

Further studies on generation of macrophages in in vitro cultures of mouse spleen cells and its inhibition by the capsular polysaccharide of Klebsiella pneumoniae.

Although the number of macrophages detected in cultures of mouse spleen cells at the start of the culture was very small, it markedly increased during further incubation. Macrophages were generated not only from the glass-adherent cell fraction of spleen cells, but also from the nonadherent cell fraction obtained after removal of adherent cells either by incubating in glass petri dishes or by passing through a glass bead column. The generation of macrophages from the nonadherent cell fraction occurred even when it was separated as late as 48 hr after the start of the culture. The phagocytic activity of macrophages newly generated from the nonadherent cell fraction was relatively weak, but it was activated during further incubation. Based on these results, the maturation process of macrophages can be divided into at least the following four stages; glass-nonadherent nonphagocytic precursor cells, glass-adherent nonphagocytic precursor cells, immature macrophages with low phagocytic activity, and mature macrophages with full phagocytic activity. The addition of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to cultures of spleen cells markedly suppressed the generation of macrophages. The suppressive effect of CPS-K depended on its dosage, and the minimum concentration of CPS-K showing a definite effect was 0.05 μg/ml. CPS-K inhibited further generation of macrophages in either the nonadherent or adherent cell fraction at any time after the start of the culture. The suppressive effect of CPS-K on the generation of macrophages could not be reversed by simple washing of spleen cells which had been kept in contact with CPS-K for 3 hr. There was no evidence which showed that CPS-K exhibited direct cytotoxic effects on spleen cells in the culture.     

The protective action of disodium cromoglycate on alveolar macrophages in phagocytosis of fibrogenic silica.

Treatment of fibrogenic silica (DQ-12) with Disodium Cromoglycate (DSCG) prior to its in-vitro and in-vivo phagocytosis by alveolar macrophages prevents the destruction of the cells and the fibrosis of lung tissue which are a consequence of phagocytosis. However, the treatment of alveolar macrophages with DSCG before phagocytosis of the silica had no, or a negligible, protective effect on the cells. Acid phosphatase activity which was significantly enhanced above the control in cells phagocytosing the silica was returned to the range found in phagocytosis of inert dust when silica treated with DSCG was phagocytized. The inhibitor of DNA- dependent RNA synthesis actinomycin D caused an increase of acid phosphatase activity. DSCG did not depress the phagocytic ability of alveolar macrophages. It appears that the catabolic enzyme process predominant in cells phagocytosing DQ-12 was under control in cells phagocytosing DQ-12 treated with DSCG and that DSCG probably acted as a regulator of the factors permitting catabolism. From the results it is suggested that the equilibrium of the enzyme reactions which accompany phagocytosis was such that the integrity of the phagocytes was preserved.     

The effect of estradiol on the cell kinetics in the uterine and cervical epithelium of neonatal mice.

The effect of estradiol-17beta on the length of the various phases of the cell cycle was studied in the neonatal mouse uterine, and cervical epithelium. A double labelling method was used, and in addition labelled mitoses were counted. In the uterus proper, estradiol shortens the length of the total cell cycle. TC, from 17-9 hr to 15-7 hr, and the duration of S phase, Ts from 6-7 to 5-1 hr 6 hr after estradiol treatment. 12 hr after estradiol treatment, TC is shortened to 7-4 hr and Ts to 4-5 hr. The shortening of TC at 12 hr is manily due to an effect on TG1, which is shortened from 8-55 hr in untreated animals to 1-8 hr in estradiol treated animals. The TC of cervix epithelium cells in untreated animals was found to be 21-8 hr. After treating the mice for 6 hr with estradiol the tc was now increased to 47 hr and further to 61-2 hr following 12 hr treatment with the hormone. Ts increases from 8-3 hr to 15-2 hr following 6 hr estradiol treatment, and to 15-4 hr after 12 hr treatment. The effect is most pronounced in TG1, which is lengthened from 10-95 hr in untreated animals to 28-1 hr and 43 hr, respectively, in animals treated for either 6 or 12 hr with estradiol.