Microbial adjuvant and autoimmunity. II. Production of lesions in mice immunized with syngeneic tissue extracts together with the capsular polysaccharide of Klebsiella pneumoniae.

The target organs of mice immunized with the respective syngeneic tissue extracts together with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a powerful adjuvant were examined for production of lesions. In 15 out of 24 mice injected three times or more with syngeneic eyeball extracts and CPS-K adjuvant at intervals approximately 30 days, severe eyeball lesions developed in which the normal structure was almost completely lost. A large part of the eyeball tissue of these mice was replaced by infiltration with cells such as lymphocytes, plasma cells and other mononuclear cells and by connective tissue. No definite eye lesions developed in mice injected with CPS-K alone, eyeball extracts alone or eyeball extracts emulsified in complete Freund's adjuvant (CFA). In all of mice injected four times with thyroid gland extracts and CPS-K at intervals of approximately 30 days, definite thyroid gland lesions were produced. In three out of five mice of this group, the thyroid lesions were so severe that the normal thyroid follicular structure was almost completely lost, and a large part of the thyroid gland was replaced by infiltration with lymphocytes, plasma cells and other mononuclear cells and in part by connective tissue. In only one out of five mice injected with thyroid gland extracts emulsified in CFA, definite but milder thyroid gland lesions developed. No definite thyroid lesions developed in the remaining four mice of this group and also in any of the mice injected with thyroid gland extracts alone or CPS-K alone. Repeated injections of lymphoid tissue extracts and CPS-K also induced pathological changes in the spleen and lymph nodes, although less marked than those in the cases of the eyes and thyroid gland. The most remarkable change was a decrease in numbers of small lymphocytes at the areas surrounding the central arterioles in the white pulp of the spleen and the post-capillary venules in the cortex of the lymph nodes. From these results it has been concluded that our system can provide new and useful models for autoimmune diseases in man.     

Microbial adjuvant and autoimmunity. III. Histological studies of development of experimental autoimmune thyroiditis in mice immunized with syngeneic thyroid extract together with the capsular polysaccharide of Klebsiella pneumoniae.

Experimental autoimmune thyroiditis could be produced in SMA, C3H/He and C57BL mice by repeated injection at intervals of 30 days of syngeneic thyroid extract mixed with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a powerful adjuvant. The sequence of the development of autoimmune thyroiditis was histologically followed using SMA strain of mice, in which the thyroid lesions were most marked. A single injection of the thyroid extract mixed with CPS-K induced interstitial infiltration of small lymphocytes and other mononuclear cells, and follicular architecture was partially damaged. At early times (5 to 12 days) after the secondary injection of the material, there were aggregation of polymorphonuclear neutrophilic leukocytes with formation of microabscesses in the follicles and hyaline degeneration of small vessels in the thyroid glands, suggesting that this stage of the thyroid lesions was expression of an Arthus reaction. The maximal severity of the thyroid lesions was reached at 30 days after the secondary injection. At this time, the thyroid lesions consisted of extensive infiltration of small lymphocytes, plasma cells and other mononuclear cells with complete loss of follicular architecture and mild proliferation of fibrous connective tissues. Even at 200 days after the secondary injection, there was no evidence of spontaneous regression and resolution of the thyroid lesions. Based on the histological findings, it seems likely that in our system cell-mediated immunity plays a dominant role in initiation and maintenance of thyroiditis and humoral antibodies do so in its aggravation.     

Microbial Adjuvant and Autoimmunity

Definite lesions in the exocrine pancreas were produced when SMA mice were immunized eight times at intervals of 30 days with a mixture of extract of pooled pancreas from syngeneic mice and the capsular polysaccharide of Klebsiella pneumoniae type 1 Kasuya strain (CPS-K), whereas no pancreatic lesions were produced in mice given CPS-K alone or pancreatic extract alone. The typical histological changes were characterized by infiltration with lymphocytes, plasma cells, and other mononuclear cells, degeneration and lysis of the acinar cells, destruction of the lobular architecture, and replacement by fatty tissue and fibrous connective tissue. The endocrine islets were well preserved. No specific histological changes were produced in the organs other than the pancreas in these mice. Most of mice immunized with pancreatic extract mixed with CPS-K produced serum precipitins to syngeneic pancreatic antigens. However, severe pancreatic lesions were also produced in mice showing no definite precipitin production.     

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.     

Characterization of autoantigens relevant to autoimmune ophthalmitis and thyroiditis in mice immunized with the syngeneic tissue extracts and Klebsiella O3 lipopolysaccharide

The histological localization and biochemical properties of the autoantigens relevant to experimental autoimmune ophthalmitis and thyroiditis were studied using sera from mice hyperimmunized with the corresponding tissue extract of syngeneic mice and Klebsiella O3 lipopolysaccharide (KO3 LPS) as a potent adjuvant. Specific antigens were detected in the lens of the eyeball by immunofluorescence test with sera from mice in which ophthalmitis had been induced and the antigens were lenticular proteins with molecular weights (MW) of 15,000 (15K) to 25K, and 45K. The lenticular proteins with MW of 15K to 25K correspond to the subunits of crystalline. These findings clearly demonstrated that our experimental model for autoimmune ophthalmitis was classified as the lens-induced uveitis. The colloids of the thyroid follicles and the follicular cells were markedly stained by sera from mice in which thyroiditis had been induced. One of the autoantigens detected in the thyroid gland was biochemically consistent with a thyroglobulin subunit. It was also shown that these autoantigens detected in the present study were organ-specific but not species-specific. The nature of autoantigens in the eye and the thyroid gland is discussed.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

Changes in the number of cells and the weight of various lymphoid organs of mice, such as the regional lymph node (right inguinal node), spleen, thymus, bone marrow, and peripheral blood, were followed after the subcutaneous injection of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K). For comparison, the changes after injection of various polyclonal lymphocyte activators (PLA) including various preparations of bacterial lipopolysaccharide (LPS) were concurrently studied. The number of cells of all of the lymphoid organs tested and that of nucleated cells in the peripheral blood decreased significantly within a few days after injection of CPS-K, and increased later. Above all, the increase in the number of cells and in the weight of the regional lymph node was most prominent (about 10 times larger than that of the normal control). Such a marked increase in the number of cells of the regional lymph node was not induced by the injection of any preparation of LPS or any other PLA tested. The initial decrease in the number of cells after CPS-K injection was most marked and long lasting in the thymus. Although LPS prepared by Westphal's method from Escherichia coli O55 or Salmonella enteritidis exhibited a stronger decreasing effect on the number of cells of the thymus, the effect of LPS prepared by Westphal's method from E. coli O111 or that by Boivin's method from E. coli O55 was similar to that of CPS-K. It is concluded therefore that CPS-K has the ability to decrease the number of cells of various lymphoid organs, especially that of the thymus, initially after injection, which is a property in common with LPS, and CPS-K has a unique ability to increase markedly the cells of various lymphoid organs, especially those of the regional lymph node, at later stages after injection. Considering that CPS-K exhibits a much stronger adjuvant effect on the antibody response than does LPS or other polyclonal lymphocyte activators, it is suggested that this extraordinarily potent activity of CPS-K in increasing the number of cells of the regional lymph node is closely related to its strong adjuvant action.     

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

A study was performed to clarify the roles of primary and secondary injections of antigen and adjuvant (capsular polysaccharide of Klebsiella pneumoniae, CPS-K) in induction of antibody responses and in development of immunological memory in mice to bovine serum albumin (BSA). A primary injecion of BSA alone neither induced significant primary antibody response nor increased immunological memory for a secondary antibody response but, if primary injections of BSA and CPS-K were performed simultaneously, high antibody responses were induced. Moreover, a prior injection of BSA alone or CPS-K alone decreased the level of primary antibody response and the degree of increase in memory following the subsequent injection of BSA mixed with CPS-K. In contrast, a secondary injection of BSA alone into mice once primed with a mixture of BSA and CPS-K elicited very high secondary type antibody response and increased secondarily the memory for a tertiary antibody response. Injection of CPS-K simultaneously with or shortly before or after the secondary injection of BSA did not increase the level of the secondary antibody response and the degree of the secondary increase in memory. Augmentation of the secondary antibody response was elicited by simultaneous injection of CPS-K only when the secondary response was induced inadequately by a suboptimum or supraoptimum dose of antigen.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The sequence of histological changes in the regional lymph node and other lymphoid organs of mice injected with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) or bacterial lipopolysaccharide (LPS) was followed. Injection of CPS-K, but not LPS, induced the following characteristic histological changes in the regional lymph node. In the early stage there was a marked decrease in the number of small lymphocytes, accompanied by the appearance of scattered fragmented nuclei and infiltration of polymorphonuclear neutrophilic leukocytes, and in the late stage there was marked proliferation of macrophage-like cells and pyroninophilic cells. Histological changes in the thymus and spleen and changes in cell populations in the bone marrow and peripheral blood after CPS-K injection were essentially the same as after LPS injection. Since CPS-K has a much stronger adjuvant action on antibody response than does LPS, it is suggested that the characteristic histological changes in the regional lymph node after injection of CPS-K are closely related to its extraordinarily strong adjuvant action.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

A study was made to characterize the active substance for the extraordinarily strong adjuvant effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) type 1 Kasuya strain. CPS-K was fractionated into acidic and neutral CPS-K by the addition of cetyl-pyridinium chloride. Neutral CPS-K exhibited an extremely strong adjuvant effect. The active substance in neutral CPS-K was precipitable when mixed with a rabbit homologous antiserum. The neutral CPS-K antigen was serologically distinct from the O antigen and from the acidic CPS-K which was the type-specific capsular antigen. Among preparations of neutral CPS-K from eight different strains of K. pneumoniae tested, the preparation from only one strain (MH-2) exhibited a strong adjuvant effect comparable to that of the neutral CPS-K from the Kasuya strain. The neutral CPS-Ks from Kasuya and MH-2 strains were antigenically identical. This antigen was not found in all preparations of neutral CPS-Ks obtained from seven different strains. Preparations of acidic CPS-Ks from all strains of K. pneumoniae tested with various serologic types including Kasuya and MH-2 strains were found to exhibit only weak adjuvant effects. The active substance (neutral CPS-K antigen from Kasuya strain) was shown to form a single peak upon analyses by gel filtration (Sephadex G-100) and ultracentrifugation. Sedimentation coefficient of the substance was approximately 20 S at a concentration of 5 mg per ml in 0.1 M NaCl. The active substance finally purified by gel filtration contained 65% sugars (as glucose equivalents), 6.8% hexuronic acids, 2.6% hexosamine, 2.3% proteins, and very small amounts of lipids.     

Antigens of Pasteurella tularensis: Preparative Procedures

Ether-water (EW) extraction of Pasteurella tularensis produced better antigens than five other chemical procedures. EW extracts produced from stationary-phase, liquid-grown, saline suspensions of strain SCHU S4 cells regularly induced agglutinin and precipitin formation in rabbits. Mice, guinea pigs, and monkeys also responded to EW extracts but with lower antibody levels. The use of strains of lower virulence, acetone-dried cells, organisms grown on a solid medium, and abbreviated extraction conditions all resulted in extracts with a diminished antigenicity, but logarithmic-phase and stationary-phase cells yielded equivalent EW extracts. The use of adjuvant, hyperimmunization, and large doses of antigen increased the precipitin responses of rabbits without appreciably altering the agglutinin response. By the appropriate combination of centrifugal fractionation of EW extracts, use of adjuvant, and vaccination schedule, rabbit antisera with either predominantly agglutinating or precipitating activities were obtained.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The neutral fraction (neutral CPS-K) of Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, has already been reported as the active substance responsible for the strong adjuvant effect of CPS-K. The present results demonstrate that neutral CPS-K exhibits further common biological activities with lipopolysaccharide (LPS) isolated from Salmonella enteritidis. The intensity of the lethality in mice of neutral CPS-K by the intraperitoneal route is very similar to that of LPS. Its lethality for mice by the intravenous (i.v.) route is significantly stronger than that of LPS, because the degree of increase in the sensitivity to their lethality by i.v. challenge is smaller for LPS than for neutral CPS-K. The intensity of the pyrogenicity of neutral CPS-K in rabbits is approximately one-tenth of that of LPS as judged by the minimal pyrogenic doses and fever indices. The skin-preparatory potency of neutral CPS-K for the dermal Shwartzman phenomenon in rabbits is also approximately one-tenth of that of LPS compared on the basis of the minimal skin-preparatory doses. When injected i.v., neutral CPS-K exhibits a provocative effect on hemorrhagic reactions in skin sites prepared with neutral CPS-K or LPS.     

Different effects of the capsular polysaccharide of Klebsiella pneumoniae on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T-dependent antigen, we studied the effects of PBA on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen. Antibody-forming cells (AFC) of IgM and IgG types were estimated as anti-SRBC direct and indirect plaque-forming cells (PFC), and the B cells with precursor activities involving generation of AFC and supplementing new B cells as rosette-forming cells (RFC) of the B-cell type. Stimulation of normal mice by CPS-K caused a definite increase in the number of direct PFC but not in that of indirect PFC and RFC in the spleens. The responsiveness of spleen cells of CPS-K-treated mice to generate PFC and RFC responses to a subsequent injection of SRBC was lower than that of CPS-K-untreated normal mice. In this case, the responsiveness to generate RFC and indirect PFC was inhibited more strongly by CPS-K than that to generate direct PFC. When CPS-K was injected into normal mice simultaneously with SRBC, CPS-K never decreased but increased the levels of PFC and RFC responses to SRBC. In the spleens of SRBC-primed mice, the number of RFC was markedly decreased following injection of CPS-K, the number of direct PFC was increased only slightly and the number of indirect PFC was increased very slightly. The responsiveness of spleen cells of these CPS-K-treated SRBC-primed mice to generate secondary PFC and RFC responses to a subsequent injection of SRBC was much lower than that of CPS-K-untreated SRBC-primed mice. In this case, the responsiveness to generate the secondary RFC and indirect PFC responses was more strongly inhibited by CPS-K than that to generate the secondary direct PFC response. When CPS-K was injected into SRBC-primed mice simultaneously with the secondary injection of SRBC, there were marked decreases in the level of the secondary RFC response and slight decreases in that of the secondary indirect PFC response, but little change in that of the secondary direct PFC response. From these results it has been concluded that CPS-K provides the positive signal (the minor action) and the negative signal (the major action) to various subpopulations of B cells functioning at various stages of the immune response to T-dependent antigen in different ways, and acts to regulate the levels of B-cell responses to the antigen-mediated positive signal.     

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.     

Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells: IV. Correlation with H-2 haplotypes

In vitro primary syngeneic sensitization on monolayers of thyroid epithelial cells was performed with 21 inbred strains of mice representing 11 original H-2 haplotypes. Significant differences in the proliferative responses, assessed by thymidine uptake, were found to be related to the major histocompatibility complex haplotype. This result was further confirmed using congenic resistant strains of mice. In comparison with the experimental autoimmune thyroiditis induced by syngeneic thyroglobulin and adjuvant, primary syngeneic sensitization on monolayers of thyroid epithelial cells appeared to be under the same genetic control (H-2^k strains being good responders, while H-2^b mice are poor responders).     

Changes in the Protein Composition of the Shoot Apical Bud of Sinapis alba in Transition to Flowering

Vegetative plants of Sinapis alba L. grown in short days were induced to flower by expsoure to one or continuous long days. In both inductive conditions, the first flowers were initiated about 60 h after the start of the treatment. Soluble protein extracts were prepared from apical buds and just-expanded leaves of both vegetative and induced plants. Rabbit antisera were prepared using extracts from vegetative and reproductive buds. Immunodiffusion tests were performed. Analysis of the precipitin bands indicated that: (1) one antigenic protein was present in the vegetative buds and disappeared from the buds of induced plants between 96 and 240 h after the start of the inductive treatment; (2) the concentration of a another antigenic protein increased in buds of induced plants 30 h after the start of the inductive treatment; (3) the concentration of a third antigenic proteín increased in buds of induced plants at 96 h.     

Microbial adjuvant and autoimmunity. IV. The induction of thyroid lesions in syngeneic X-irradiated mice by the transfer of spleen cells from mice immunized with thyroid extract and Klebsiella O3 lipopolysaccharide

The role of humoral and cellular immune responses in the initiation and maintenance of autoimmune thyroiditis was investigated in mice immunized with syngeneic thyroid extract and Klebsiella O3 lipopolysaccharide (KO3 LPS) as an adjuvant. The transfer of spleen cells from hyperimmunized mice to 400R-irradiated syngeneic mice produced definite lesions in the thyroid glands, whereas the transfer of immune sera failed to do so. No lesions were induced in normal intact mice by the same transfer of sera and spleen cells from hyperimmunized mice. It was suggested that the induction of thyroiditis by immunization using KO3 LPS adjuvant is primarily due to cell-mediated immunity and that pretreatment of mice by X-irradiation is essential for production of the lesions. The role of X-irradiation in the induction of thyroiditis was discussed.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. II. Influence of time after BCG inoculation on production of interferon and cytotoxin by capsular polysaccharide of Klebsiella pneumoniae or by bacterial lipopolysaccharide and on hyperreactivity to their lethal effects.

The time course of the occurrence of hyperreactivity in interferon and cytotoxin responses to the active substance (neutral fraction) of the capsular polysaccharide of Klebsiella pneumoniae (neutral CPS-K) and bacterial lipopolysaccharide (LPS) and of the hyperreactivity to their lethal effects was followed after infection with BCG in SMA and ICR strains of mice. The duration of these hyperreactivities of BCG-infected mice depended on the inoculum doses of BCG. The time patterns of the hyperreactivity to the lethal effects of neutral CPS-K and LPS were similar in both strains of mice, although the maximum toxicity of LPS by the intraperitoneal route in BCG-infected mice on a weight basis was stronger than that of neutral CPS-K. Irrespective of inducer and mouse strain, the time pattern of the hyperreactivity to produce cytotoxin was similar to that of the hyperreactivity to produce interferon. The patterns for these phenomena when neutral CPS-K was used as an inducer were also similar to those when LPS was used. In ICR mice the hyperreactivity in interferon and cytotoxin responses to either neutral CPS-K or LPS decayed significantly earlier than the hyperreactivity to their lethal effects, whereas in SMA mice the occurrence of both types of hyperreactivities seemed to be associated. Therefore, it is suggested that the mechanism for the hyperreactivity in interferon and cytotoxin responses to neutral CPS-K or LPS in BCG-infected mice is not necessarily the same as that for the hyperreactivity to their lethal effects.     

Formation of Mononuclear Phagocyte (Macrophage) Colonies by Mouse Spleen Cells in Liquid Culture

The effect of the capsular polysaccharide of Klebsiella pneumoniae type 1 Kasuya strain (CPS-K) on the formation of macrophage colonies in cultures of mouse spleen cells was investigated by the liquid culture technique during an incubation period of 7–8 days. CPS-K markedly inhibited further generation of macrophage colonies when added at any time after the beginning of culture, whereas it showed no destructive effect on macrophage colonies which were already formed before its addition. When CPS-K was present throughout the incubation period, such a low concentration as 0.05 μg/ml significantly inhibited colony formation, and the intensity of its inhibitory effect depended on its dose in the range of 0.005–50 μg/ml. The inhibitory effect persisted even if CPS-K was washed out after spleen cells were kept in contact with 20 μg of CPS-K per ml at 37 C for 6 hr. It was found that the inhibitory effect of CPS-K on colony formation was not mediated through its action on T cells, B cells or macrophages, and that it was not due to the generation of suppressor cells capable of inhibiting colony formation. It is concluded therefore that CPS-K directly inhibits the proliferation of macrophage colony-forming cells. The active substance responsible for the inhibitory effect of CPS-K on colony formation is the neutral polysaccharide fraction of CPS-K.     

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.