Immunology 1976 30 229
Cellular aspects of non-specific stimulation of antibody production by capsular polysaccharide of Klebsiella pneumoniae
I.
NAKASHIMA, T. KOJIMA & N. KATO Department of Bacteriology, Nagoya University School of Medicine, Showa-ku, Nagoya, Japan
Received 4 June 1975; acceptedfor publication 6 August 1975
Sumwmry. Comparative studies were made of the increase in the numbers of plaque-forming cells (PFC), rosette-forming cells (RFC) and haemolytic foci for erythrocyte antigens in the spleens of mice given a non-specific stimulus (the capsular polysaccharide of Klebsiellapneumoniae (CPS-K)) and an antigenic stimulus (sheep red blood cells (SRBC)). The number of direct PFC for SRBC was increased by injection of CPS-K to as high a level as that obtained by injection of SRBC. In contrast, by injection of CPS-K the numbers of indirect PFC, RFC (probably the antibody-forming cell precursors) and haemolytic foci were not increased significantly, whereas all of them were increased markedly by injection of SRBC. The maximal number of PFC in mice injected with CPS-K was approximated to the number of background RFC of the same mice. Injection of CPS-K generated 25-130 times more direct PFC for each of three kinds of erythrocyte antigens, SRBC, rabbit red blood cells and chick red blood cells, than background PFC, whereas the total number of spleen cells was not increased significantly or increased very slightly. Repeated injections of CPS-K were not significantly more effective for increase in the number of direct PFC than a single injection of CPS-K. Injection of CPS-K could generate many direct PFC in mice which had
been thymectomized, irradiated and reconstituted with foetal liver cells. In mice injected with CPS-K, increase in (or maintenance of) the numbers of direct PFC and RFC were inhibited by injection of a mitogen inhibitor, vinblastine sulphate, but their sensitivities to the drug were less than those found in mice immunized with SRBC. It has been concluded from these results that in mice injected with CPS-K a large number of antibody-forming cell precursors are differentiated to direct PFC through one division or a few divisions of the individual cells, and that the inability of CPS-K to induce sufficient cell divisions of the individual precursor cells is the cause of the lack of increase in the number of indirect PFC and in immunological memory for secondary PFC responses in mice injected with CPS-K.
INTRODUCTION It has been recently shown that antibody-forming cells (AFC) are generated from their precursors in vitro not only through the action of specific antigens but also through the action of non-specific mitogenic substances, such as bacterial lipopolysaccharide (LPS) (Andersson, Sjoberg and Moller, 1972), purified protein derivative of tuberculin (PPD) (Nilsson, Sultzer and Bullock, 1973), pokeweed mitogen (Parkhouse, Janossy and Greaves, 1972) and dextran sulphate (Diamantstein, RuhO, Vogt and
Correspondence: Dr I. Nakashima, Department of Bacteriology, Nagoya University School of Medicine, Showaku, Nagoya 466, Japan.
229
230
I. Nakashima, T. Kojima & N. Kato
Bochert, 1973). The action of LPS to generate AFC is usually very weak in vivo (Andersson et al., 1972). We have shown recently, however, that injection of relatively large doses of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) type 1 Kasuya strain induces in mice a marked increase in IgM type antibodies to various antigens which is comparable to the increase in IgM type antibodies produced by specific antigenic stimuli (Nakashima & Kato, 1974). In general, AFC generated by non-specific stimuli are exclusively of IgM type (Andersson et al., 1972; Parkhouse et al., 1972; Diamantstein et al., 1973; Nilsson et al., 1973; Nakashima and Kato, 1974). Definite immunological memory is not induced by non-specific stimuli (Nakano, Uchiyama, Tanabe and Saito, 1975; Nakashima and Kato, 1974). AFC can be generated by non-specific stimuli without the help of such accessory cells as T cells (Greaves, Owen and Raff, 1973; Coutinho and Moller, 1975). The lag period for the increase in AFC by non-specific stimuli is shorter than that by specific stimuli (Nakano et al., 1975; Coutinho and Moller, 1975). However, the true cytodynamics of generation of AFC by non-specific stimuli are not yet made clear. A number of the so-called B-cell mitogens have been shown to stimulate differentiation of the antibody-forming cell precursors (AFCP) to direct plaque-forming cells (PFC), although different mitogenic substances may affect different subpopulations of B cells (Coutinho and Moller, 1975). Nevertheless few studies have been made to answer the question of whether mitogenic substances would induce an increase in rosette-forming cells (RFC) (probably AFCP), and of whether mitogenic substances would induce the appearance of haemolytic foci which is regarded as the evidence for the clonal proliferation of AFC or AFCP. The purpose of the present study is to make clear the cytodynamics of generation of AFC and AFCP following the nonspecific stimulus of CPS-K in an in vivo system. We have conducted comparative studies on the kinetics of increase in direct and indirect PFC, RFC and haemolytic foci for erythrocyte antigens by single or repeated injections of erythrocytes or CPS-K.
MATERIALS AND METHODS Animals Male and female SMA mice approximately 6 weeks of age were used.
Erythrocyte antigens and capsular polysaccharide of Klebsiella pneumoniae (CPS-K) Sheep red blood cells (SRBC), rabbit red blood cells (RRBC) and chick red blood cells (CRBC) were used as antigens. CPS-K was prepared from the culture fluid of K. pneumoniae type 1 Kasuya strain as described previously (Nakashima, Kobayashi and Kato, 1971). Each substance was injected into mice intramuscularly (i.m.) or intraperitoneally (i.p.). In some experiments, it was injected directly into the spleen by laparotomy.
Preparation of cell suspensions Mouse spleens were dissected to prepare single cell suspensions in Eagle's minimum essential medium (MEM). They were washed once with MEM by centrifugation at 800 r.p.m. for 7 min. Cell numbers were measured with a haemocytometer. Estimation of plaque-forming cells (PFC) AFC were enumerated as PFC using the technique of Cunningham and Szenberg (1968). For direct assay (estimation of PFC of IgM type), Cunningham and Szenberg's microslide chambers were filled with a mixture of 0 015 ml of a 50 per cent suspension of erythrocytes, 0015 ml of guinea-pig complement and 0-1 ml of spleen cell suspension. For indirect assay (estimation of PFC of IgG type), 0 005 ml of rabbit antiserum to mouse IgG (Nakashima and Kato, 1974) diluted 1:40 was added to this mixture. The chambers were incubated for 1 5 h at 370 for development of haemolytic plaques. The number of indirect PFC was determined by subtracting the number of direct PFC from the total number of PFC determined by addition of antiserum to mouse IgG. In some experiments, mixtures of two kinds of erythrocytes were used as indicator cells. In this case, incomplete haemolytic plaques were enumerated as PFC secreting antibody to either of the two indicator cells. The shape and size of erythrocytes of the three kinds (SRBC, RRBC and CRBC) were different from each other. The cell type lysed in incomplete haemolytic plaques could therefore be examined under a microscope. Estimation of rosette-forming cells (RFC) The suspension-centrifugation technique of McConnell, Munro, Gurner and Coombs (1969) was used. A mixture of 005 ml of spleen cell suspension (approximately 5 x 107/ml), 0-1 ml of a suspension of indicator erythrocytes and 0-15 ml of phosphate-
Antibody synthesis stimulation by CPS-K buffered saline at pH 7-2 (PBS) was centrifuged at 500 r.p.m. for 5 min and stored at 40 for at least 20 min before enumeration of RFC. The deposited cells were resuspended with a pipette and placed on a haemocytometer. Approximately 1.5 x 105 spleen cells were scanned and numbers of cells forming typical rosette or morula (lymphocytes binding more than approximately ten erythrocytes) were counted. It has been shown that RFC binding more than ten erythrocytes are RFC of B-cell type (probably AFCP), whereas RFC binding fewer erythrocytes may be RFC of T cell type (Haskill, Elliot, Kerbel, Axelrad and Eidinger, 1972; Charreire, Dardenne and Bach, 1973; Warner, 1974). However, macrophages can also form rosettes (Charreire, Dardenne and Bach, 1973). We tested if any significant numbers of rosette-forming macrophage contaminated the RFC population. Normal mice or mice which had been injected with CPS-K or SRBC were injected intravenously (i.v.) with colloidal carbon (25 mg/100 g) 2 h before being killed. Most of the carbonlabelled RFC, however, bound fewer erythrocytes than ten. The carbon-labelled RFC which bound more than ten erythrocytes bound some leucocytes together with erythrocytes. Only a small percentage, if any, of the RFC which we estimated appear therefore to be macrophages. In addition, in a study in which spleen cells of normal mice or CPS-K-treated mice were mixed with two different kinds of erythrocytes such as SRBC and CRBC, no significant numbers of RFC were found to bind both kinds of erythrocytes. This also indicates that most of RFC which we estimated are antigen-specific.
231
Estimation of haemolytic foci The haemolytic focus was assayed by the technique of Playfair, Papermaster and Cole (1965). Spleens were minced by a razor into pieces smaller than 1 mm3 and sequentially transferred with forceps into the agarose plate, which had been made by solidifying a mixture of 0 05 ml of a suspension of 50 per cent erythrocytes, 0-2 ml of x 2 MEM and 0-2 ml of 1 0 per cent agarose on basal 0 7 per cent agarose in MEM. These plates were incubated at 370 for 1 h. After removing the pieces of the spleen, they were incubated at 370 for 30 min with guinea-pig complement diluted 1:10. We scored as haemolytic foci the areas of lysis that were larger than 0-5 mm in diameter. Thymectomy and irradiation Adult mice were thymectomized by the technique of Miller (1960). Intact or thymectomized mice were exposed to total-body irradiation of 800 R Co60. In the case of thymectomized mice, the irradiation was performed 3-5 weeks after thymectomy. The irradiated mice were protected with i.v. injection of various numbers of spleen cells or 5 x 106 foetal liver cells from syngeneic mice approximately 20 h after the irradiation. Mitosis inhibitor Vinblastine sulphate (Eli Lilly Company, Indianapolis, Indiana) was used as a mitosis inhibitor (Pazdernik and Uyeki, 1973). RESULTS
Comparative examination of PFC and RFC A new method was used to study if and how PFC populations overlap with RFC populations. The spleen cell suspension (0-05 ml) was mixed with 0-015 ml of a 20 per cent suspension of SRBC and 0-015 ml of guinea-pig complement. The mixture was centrifuged at 500 r.p.m. for 5 min, resuspended and introduced into Cunningham's chamber, of which the underlying microslide was sectioned into 1 x 1 mm space by engraving lines with a diamond pen. These chambers were settled at 40 more than 20 min at a horizontal position and the number and position of RFC were recorded under a microscope. The chambers were then incubated at 370 for 30 min, and again examined for the number and position of RFC and PFC.
Kinetics of increase in the numbers of PFC and RFC for SRBC in the spleens of mice injected with SRBC or CPS-K A kinetic study was made of the increase in the numbers of direct PFC, indirect PFC and RFC for SRBC in the spleens following injection of SRBC or CPS-K (Fig. 1). In the spleens of normal untreated mice, there were small numbers of background PFC and large numbers of background RFC. The numbers of both direct PFC and RFC were found to be increased by 3 days after i.p. injection of SRBC. The highest levels of direct PFC and RFC were reached 4 or 5 days after injection, and their levels decreased thereafter. The number of indirect PFC was found to be increased by 5 days after injection of SRBC,
232
2L Nakashima, T. Kojima & N. Kato 1000
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Figure 1. Increase in direct PFC, indirect PFC and RFC for SRBC in the spleens of mice injected with SRBC or CPS-K. Mice were injected i.p. with 2x 108 SRBC (x), i.m. with 2 x 108 SRBC (A) or i.m. with 1000 pg of CPS-K (o). Each point represents ) or total numbers/spleen (- - - -) (x 10-3) of six to twelve mice with the geometric mean of numbers per 106 spleen cells ( its standard deviation.
the highest level was reached at 10 days, and its level decreased thereafter. After i.m. injection of SRBC, the number of direct PFC, indirect PFC and RFC were increased in similar fashions to after i.p. injection, although their peak levels were much lower. On the other hand, the number of direct PFC increased more rapidly after i.m. injection of CPS-K than after i.m. or i.p. injection of SRBC. The peak level of direct PFC after i.m. injection of CPS-K was much higher than that after i.m. injection of SRBC, although it was lower than the peak level after i.p. injection of SRBC. The number of direct PFC in the spleens of mice injected with CPS-K decreased almost as rapidly as those in the spleens of mice injected with SRBC. Despite such a marked increase in the numbers of direct PFC, the numbers of indirect PFC and RFC were not increased significantly after injection of CPS-K. The peak level of direct PFC in the spleens of mice injected with CPS-K was approximately the same as the level of RFC in the same mice. Relationship between direct PFC and RFC populations in the spleens of mice injected with CPS-K From the experimental results that in the spleens of mice injected with CPS-K the number of direct PFC at the peak approximated the number of RFC, a question arose as to whether direct PFC generated after CPS-K injection and RFC belonged to distinct or the same cell populations. The positions of the individual PFC and RFC, which were developed in
the same Cunningham's chamber, were recorded before and after incubation of the chamber (Table 1). Some RFC disappeared after incubation for 1 h at 370, whereas small numbers of RFC appeared at the new positions after incubation. These phenomena may be related to the appearance and disappearance of receptors for SRBC on the cell surface of lymphocytes (Warner, 1974) during incubation. On the other hand, only small numbers of direct PFC appeared at the position where an RFC was recorded before incubation. Most of the direct PFC appeared at the position unrelated to the position of RFC. These results indicate that most, if not all, of the RFC and direct PFC are distinct cell populations. This finding agrees with that of Wilson (1971) that PFC in the spleens of mice immunized with SRBC had no antigen-binding receptors demonstrable at 4°. Increase in the number of PFC for various kinds of erythrocytes in the spleens of mice injected with CPS-K and its correlation with increase in the total number of spleen cells In the experiments described so far, PFC and RFC for SRBC were examined. A study was then made to see whether PFC and RFC for other kinds of erythrocytes would also be increased by injection of CPS-K (Table 2). There were different numbers of background PFC and RFC for SRBC, RRBC and CRBC in normal untreated mice. The number of background RFC for CRBC was more than that for SRBC, whereas the number of background RFC for
Antibody synthesis stimulation by CPS-K
233
Table 1. Distinction between populations of PFC and RFC in the spleens of mice injected with CPS-K*
RFC numbers/chamber
Experiment number
Time of examination
I
Before incubation After incubation
2
32 13 At the positions where RFC was detected before incubation 9 21 12 At the positions where RFC was detected before incubation 11
Before incubation After incubation
PFC numbers/chamber 0 14
At the positions where RFC was detected before incubation 2 0 2 At the positions where RFC was detected before incubation 0
At new positions 4
At new positions 1
At new positions 12 At new positions 2
* For details see Materials and Methods section. Spleens were obtained from mice 3 days after the direct injection of 100 pg of CPS-K into the spleen. Table 2. Increase in the numbers of PFC and RFC for various kinds of erythrocytes and the total numbers of cells in the spleen of mice injected with CPS-K
Experiment Materials* number injected
1
3
3
88
102 3
None CPS-K
58
53
None CPS-K
4
2
None CPS-K
Spleen cell numbers (x 10-6) 71
None CPS-K
2
Days after injection
136
63 4
120
PFC and RFC numbers
Target
PFC/spleen
PFC/106 cells
RFC/spleen
RFC/106 cells
< 50 < 50
270+ 730 1360+410 313+270 3400+ 200 < 50 < 50 360+ 90 2440+ 90 765+ 490 23,700+ 7700 < 50 760+ 580 765+ 405 5100+ 500 2970+ 810 49,000+ 9500 112+22 335+ 155
1*0 > 2-0 None
CPS-K
SRBC
115 720 350 68 6950 1920 6350 1300 1180 550 55 580
0 2
0 0
0 0
6 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
* For details see Materials and Methods section. t Mice were injected i.p. with 100 pg of CPS-K or 5x 107 SRBC 12 days after i.v. injection of foetal liver cells. PFC and haemolytic foci were determined 4 days after injection of CPS-K or SRBC.
Increase in the numbers of direct PFC and haemolytic foci for SRBC by injection of SRBC or CPS-K in the spleens of mice thymectomized, irradiated and reconstituted with foetal liver cells A study was then made to see if and how injection of SRBC or CPS-K would increase the numbers of direct PFC and haemolytic foci in the absence of T cells (Table 6). There were considerable numbers of background PFC in the spleens of mice which were thymectomized, irradiated and reconstituted with foetal liver cells. Injection of SRBC into these mice increased only slightly the number of direct PFC. In contrast, much larger numbers of direct PFC were generated after injection of CPS-K. No large haemolytic foci appeared in the spleens of these mice, although some small haemolytic foci appeared in the spleens of some control mice and mice injected with CPS-K. These results indicate that in the absence of T cells CPS-K directly affects AFCP (B cells), whereas SRBC cannot do this. Moreover, it is suggested that the helper action of T cells is essential for the development of haemolytic foci.
Dependence on cell division of the increase in the numbers of direct PFC and RFC in the spleens of mice injected with CPS-K or SRBC To see if and how the increase in the numbers of
direct PFC and RFC in the spleen by injection of CPS-K or SRBC depends upon cell division, the sensitivities to vinblastine sulphate of total spleen cells, PFC and RFC in mice injected with them and in untreated control mice were determined (Table 7). The sensitivities of these cells to the drug are expressed as the ratios of the values for mice treated with the drug and for mice not treated. A single injection of vinblastine sulphate into normal control mice decreased the numbers of total spleen cells and background PFC and RFC for SRBC. The degree of decrease for background PFC was lower than that for total spleen cells, whereas the degree of decrease for background RFC was similar to that for total spleen cells. These effects of vinblastine sulphate on the normal spleens are perhaps related to its nonspecific cytotoxic action rather than to its mitosis inhibitory action. Because the total number of spleen cells was changed by administration of vinblastine sulphate, the sensitivities to the drug of PFC and RFC for different groups of mice should be compared by use of the values per 106 spleen cells. From these results, increase in the numbers of direct PFC and RFC by injection of SRBC appeared to be markedly dependent on cell divisions. Increase in the number of direct PFC by injection of CPS-K appeared to be definitely dependent on cell divisions. The number of RFC was increased only very slightly by
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injection of CPS-K, but cell divisions appeared to be required to some extent for maintenance of RFC in these mice. Increase in the total number of spleen cells appeared also to be definitely dependent on cell divisions, suggesting that a large part of total spleen cells are affected by mitogenic action of CPS-K. The dependency on cell divisions of development of PFC in these mice appeared to be slightly higher than that of increase in total spleen cells. On the other hand, the dependency on cell divisions of development of RFC appeared to be lower than that of increase in total spleen cells. These results indicate that cell divisions are required for the increase in the number of direct PFC and for maintenance of RFC in mice injected with CPS-K, and that CPS-K induces proliferation and differentiation of different subpopulations of spleen cells to different degrees. DISCUSSION In the spleens of mice, IgM AFC for SRBC antigen are generated by the non-specific stimulus (CPS-K) almost as markedly as by the specific antigenic stimulus (SRBC). With the use of this experimental system, we compared the modes of increase in IgM AFC and IgG AFC and their precursors by the
specific and non-specific stimuli. Evidence has been presented to support the idea that increase in the number of IgM AFC (direct PFC) in the spleen of mice injected with CPS-K does not result from very frequent divisions of individual AFC or their precursors. First, injection of CPS-K did not increase the number of haemolytic foci (Table 5), which would appear through clonal proliferation of AFC or their precursors (Playfair et al., 1965). It may therefore be that injection of CPS-K generates direct PFC diffusely from relatively large numbers of the precursor cells through a few or no cell divisions, although another possibility remains to be excluded that dividing cells stimulated by SRBC stay together in one place but cells stimulated by CPS-K do not. Secondly, the number of total spleen cells was not increased greatly by injection of CPS-K, while the number of direct PFC for various antigens was increased markedly (Table 2). It appears that CPS-K, like other B-cell mitogens (Coutinho and Moller, 1975), stimulates B cells, which can be regarded as AFCP for numerous antigens and occupy approximately 50 per cent of total spleen cells (Greaves et al., 1973), non-speci-
fically to generate AFC without the help of action of T cells (Table 6). It has not been estimated what percentages of B cells in the spleens are stimulated by CPS-K. It may be reasonable, however, to consider that the total spleen cells would have increased more markedly if considerable numbers of B cells divide very frequently. Thirdly, we found that injection of CPS-K increased the number of RFC only very slightly (Fig. 1, Tables 2 and 4). It appears that most of RFC which we estimated in the present study are B cells and therefore AFCP, although the possibility that a small proportion of them may be T cells, macrophages and AFC themselves could not be completely ruled out (see Materials and Methods section and Table 1). This may therefore indicate that CPS-K does not induce marked proliferation of AFCP (B cells). This lack of stimulatory effect of CPS-K on the proliferation of RFC is possibly related to the fact that increase in immunological memory is not induced in mice iniected with CPS-K (Table 4) It has been recently shown that AFC can be generated from their precursors without cell divisions (Marbrook and Haskill, 1974; Sulitzeanu et al., 1973; Andersson and Melchers, 1974), while other investigators showed that at least one cell division is required for the generation of AFC from their precursors (Nakamura, Segal, Globerson and Feldman, 1972). In the present study, it has been shown that cell division is probably required for the generation of AFC from the precursors by CPS-K (Table 7). In mice injected with CPS-K direct PFC are increased to as high levels as background RFC (Fig. 1 and Table 2). A possible mechanism of the generation of AFC by the non-specific stimulus of CPS-K is that a large number of background RFC are stimulated by CPS-K to differentiate to direct PFC through one cell division or a few divisions. CPS-K causes no definite changes in RFC levels, but cell division is required to some extent for the maintenance of RFC at the same level as background RFC (Table 7). It may therefore be that RFC are replaced by newly differentiating precursors but the rate of generation of new RFC do not exceed that of differentiation of RFC to PFC. The alternative possibility may exist, however, that RFC respond to SRBC and a T-cell factor only and direct PFC stimulated by CPS-K come from the other cell population than RFC. Only very small numbers of IgG AFC (indirect PFC) were generated after a single injection of CPS-K, even after repeated injections (Fig. 1 and
Antibody synthesis stimulation by CPS-K Table 4). It has been reported that there are only small numbers of precursor cells for IgG AFC in untreated mice (Warner, 1974). For the marked increase in IgG PFC, therefore, the precursor cells may have to divide very frequently. Alternatively, if the switch over from IgM AFC (or their precursors) to IgG AFC (or their precursors) is the case, it may be accomplished only through frequent cell divisions of the precursor cells. In any case, the lack of frequent cell divisions of AFCP in the spleens of mice injected with CPS-K may be the cause of the fact that IgG PFC are not increased by the non-specific stimulus of CPS-K. A hypothetical model which would summarize the results in this work is shown in Fig. 2. It may be that the stem cells (or immature B cells) generate RFC (there may also be stepwise functional maturation in subpopulations of RFC) and some of the mature RFC differentiate to direct PFC in the microenvironR
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Cellular aspects of non-specific stimulation of antibody production by capsular polysaccharide of Klebsiella pneumoniae
I.
NAKASHIMA, T. KOJIMA & N. KATO Department of Bacteriology, Nagoya University School of Medicine, Showa-ku, Nagoya, Japan
Received 4 June 1975; acceptedfor publication 6 August 1975
Sumwmry. Comparative studies were made of the increase in the numbers of plaque-forming cells (PFC), rosette-forming cells (RFC) and haemolytic foci for erythrocyte antigens in the spleens of mice given a non-specific stimulus (the capsular polysaccharide of Klebsiellapneumoniae (CPS-K)) and an antigenic stimulus (sheep red blood cells (SRBC)). The number of direct PFC for SRBC was increased by injection of CPS-K to as high a level as that obtained by injection of SRBC. In contrast, by injection of CPS-K the numbers of indirect PFC, RFC (probably the antibody-forming cell precursors) and haemolytic foci were not increased significantly, whereas all of them were increased markedly by injection of SRBC. The maximal number of PFC in mice injected with CPS-K was approximated to the number of background RFC of the same mice. Injection of CPS-K generated 25-130 times more direct PFC for each of three kinds of erythrocyte antigens, SRBC, rabbit red blood cells and chick red blood cells, than background PFC, whereas the total number of spleen cells was not increased significantly or increased very slightly. Repeated injections of CPS-K were not significantly more effective for increase in the number of direct PFC than a single injection of CPS-K. Injection of CPS-K could generate many direct PFC in mice which had
been thymectomized, irradiated and reconstituted with foetal liver cells. In mice injected with CPS-K, increase in (or maintenance of) the numbers of direct PFC and RFC were inhibited by injection of a mitogen inhibitor, vinblastine sulphate, but their sensitivities to the drug were less than those found in mice immunized with SRBC. It has been concluded from these results that in mice injected with CPS-K a large number of antibody-forming cell precursors are differentiated to direct PFC through one division or a few divisions of the individual cells, and that the inability of CPS-K to induce sufficient cell divisions of the individual precursor cells is the cause of the lack of increase in the number of indirect PFC and in immunological memory for secondary PFC responses in mice injected with CPS-K.
INTRODUCTION It has been recently shown that antibody-forming cells (AFC) are generated from their precursors in vitro not only through the action of specific antigens but also through the action of non-specific mitogenic substances, such as bacterial lipopolysaccharide (LPS) (Andersson, Sjoberg and Moller, 1972), purified protein derivative of tuberculin (PPD) (Nilsson, Sultzer and Bullock, 1973), pokeweed mitogen (Parkhouse, Janossy and Greaves, 1972) and dextran sulphate (Diamantstein, RuhO, Vogt and
Correspondence: Dr I. Nakashima, Department of Bacteriology, Nagoya University School of Medicine, Showaku, Nagoya 466, Japan.
229
230
I. Nakashima, T. Kojima & N. Kato
Bochert, 1973). The action of LPS to generate AFC is usually very weak in vivo (Andersson et al., 1972). We have shown recently, however, that injection of relatively large doses of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) type 1 Kasuya strain induces in mice a marked increase in IgM type antibodies to various antigens which is comparable to the increase in IgM type antibodies produced by specific antigenic stimuli (Nakashima & Kato, 1974). In general, AFC generated by non-specific stimuli are exclusively of IgM type (Andersson et al., 1972; Parkhouse et al., 1972; Diamantstein et al., 1973; Nilsson et al., 1973; Nakashima and Kato, 1974). Definite immunological memory is not induced by non-specific stimuli (Nakano, Uchiyama, Tanabe and Saito, 1975; Nakashima and Kato, 1974). AFC can be generated by non-specific stimuli without the help of such accessory cells as T cells (Greaves, Owen and Raff, 1973; Coutinho and Moller, 1975). The lag period for the increase in AFC by non-specific stimuli is shorter than that by specific stimuli (Nakano et al., 1975; Coutinho and Moller, 1975). However, the true cytodynamics of generation of AFC by non-specific stimuli are not yet made clear. A number of the so-called B-cell mitogens have been shown to stimulate differentiation of the antibody-forming cell precursors (AFCP) to direct plaque-forming cells (PFC), although different mitogenic substances may affect different subpopulations of B cells (Coutinho and Moller, 1975). Nevertheless few studies have been made to answer the question of whether mitogenic substances would induce an increase in rosette-forming cells (RFC) (probably AFCP), and of whether mitogenic substances would induce the appearance of haemolytic foci which is regarded as the evidence for the clonal proliferation of AFC or AFCP. The purpose of the present study is to make clear the cytodynamics of generation of AFC and AFCP following the nonspecific stimulus of CPS-K in an in vivo system. We have conducted comparative studies on the kinetics of increase in direct and indirect PFC, RFC and haemolytic foci for erythrocyte antigens by single or repeated injections of erythrocytes or CPS-K.
MATERIALS AND METHODS Animals Male and female SMA mice approximately 6 weeks of age were used.
Erythrocyte antigens and capsular polysaccharide of Klebsiella pneumoniae (CPS-K) Sheep red blood cells (SRBC), rabbit red blood cells (RRBC) and chick red blood cells (CRBC) were used as antigens. CPS-K was prepared from the culture fluid of K. pneumoniae type 1 Kasuya strain as described previously (Nakashima, Kobayashi and Kato, 1971). Each substance was injected into mice intramuscularly (i.m.) or intraperitoneally (i.p.). In some experiments, it was injected directly into the spleen by laparotomy.
Preparation of cell suspensions Mouse spleens were dissected to prepare single cell suspensions in Eagle's minimum essential medium (MEM). They were washed once with MEM by centrifugation at 800 r.p.m. for 7 min. Cell numbers were measured with a haemocytometer. Estimation of plaque-forming cells (PFC) AFC were enumerated as PFC using the technique of Cunningham and Szenberg (1968). For direct assay (estimation of PFC of IgM type), Cunningham and Szenberg's microslide chambers were filled with a mixture of 0 015 ml of a 50 per cent suspension of erythrocytes, 0015 ml of guinea-pig complement and 0-1 ml of spleen cell suspension. For indirect assay (estimation of PFC of IgG type), 0 005 ml of rabbit antiserum to mouse IgG (Nakashima and Kato, 1974) diluted 1:40 was added to this mixture. The chambers were incubated for 1 5 h at 370 for development of haemolytic plaques. The number of indirect PFC was determined by subtracting the number of direct PFC from the total number of PFC determined by addition of antiserum to mouse IgG. In some experiments, mixtures of two kinds of erythrocytes were used as indicator cells. In this case, incomplete haemolytic plaques were enumerated as PFC secreting antibody to either of the two indicator cells. The shape and size of erythrocytes of the three kinds (SRBC, RRBC and CRBC) were different from each other. The cell type lysed in incomplete haemolytic plaques could therefore be examined under a microscope. Estimation of rosette-forming cells (RFC) The suspension-centrifugation technique of McConnell, Munro, Gurner and Coombs (1969) was used. A mixture of 005 ml of spleen cell suspension (approximately 5 x 107/ml), 0-1 ml of a suspension of indicator erythrocytes and 0-15 ml of phosphate-
Antibody synthesis stimulation by CPS-K buffered saline at pH 7-2 (PBS) was centrifuged at 500 r.p.m. for 5 min and stored at 40 for at least 20 min before enumeration of RFC. The deposited cells were resuspended with a pipette and placed on a haemocytometer. Approximately 1.5 x 105 spleen cells were scanned and numbers of cells forming typical rosette or morula (lymphocytes binding more than approximately ten erythrocytes) were counted. It has been shown that RFC binding more than ten erythrocytes are RFC of B-cell type (probably AFCP), whereas RFC binding fewer erythrocytes may be RFC of T cell type (Haskill, Elliot, Kerbel, Axelrad and Eidinger, 1972; Charreire, Dardenne and Bach, 1973; Warner, 1974). However, macrophages can also form rosettes (Charreire, Dardenne and Bach, 1973). We tested if any significant numbers of rosette-forming macrophage contaminated the RFC population. Normal mice or mice which had been injected with CPS-K or SRBC were injected intravenously (i.v.) with colloidal carbon (25 mg/100 g) 2 h before being killed. Most of the carbonlabelled RFC, however, bound fewer erythrocytes than ten. The carbon-labelled RFC which bound more than ten erythrocytes bound some leucocytes together with erythrocytes. Only a small percentage, if any, of the RFC which we estimated appear therefore to be macrophages. In addition, in a study in which spleen cells of normal mice or CPS-K-treated mice were mixed with two different kinds of erythrocytes such as SRBC and CRBC, no significant numbers of RFC were found to bind both kinds of erythrocytes. This also indicates that most of RFC which we estimated are antigen-specific.
231
Estimation of haemolytic foci The haemolytic focus was assayed by the technique of Playfair, Papermaster and Cole (1965). Spleens were minced by a razor into pieces smaller than 1 mm3 and sequentially transferred with forceps into the agarose plate, which had been made by solidifying a mixture of 0 05 ml of a suspension of 50 per cent erythrocytes, 0-2 ml of x 2 MEM and 0-2 ml of 1 0 per cent agarose on basal 0 7 per cent agarose in MEM. These plates were incubated at 370 for 1 h. After removing the pieces of the spleen, they were incubated at 370 for 30 min with guinea-pig complement diluted 1:10. We scored as haemolytic foci the areas of lysis that were larger than 0-5 mm in diameter. Thymectomy and irradiation Adult mice were thymectomized by the technique of Miller (1960). Intact or thymectomized mice were exposed to total-body irradiation of 800 R Co60. In the case of thymectomized mice, the irradiation was performed 3-5 weeks after thymectomy. The irradiated mice were protected with i.v. injection of various numbers of spleen cells or 5 x 106 foetal liver cells from syngeneic mice approximately 20 h after the irradiation. Mitosis inhibitor Vinblastine sulphate (Eli Lilly Company, Indianapolis, Indiana) was used as a mitosis inhibitor (Pazdernik and Uyeki, 1973). RESULTS
Comparative examination of PFC and RFC A new method was used to study if and how PFC populations overlap with RFC populations. The spleen cell suspension (0-05 ml) was mixed with 0-015 ml of a 20 per cent suspension of SRBC and 0-015 ml of guinea-pig complement. The mixture was centrifuged at 500 r.p.m. for 5 min, resuspended and introduced into Cunningham's chamber, of which the underlying microslide was sectioned into 1 x 1 mm space by engraving lines with a diamond pen. These chambers were settled at 40 more than 20 min at a horizontal position and the number and position of RFC were recorded under a microscope. The chambers were then incubated at 370 for 30 min, and again examined for the number and position of RFC and PFC.
Kinetics of increase in the numbers of PFC and RFC for SRBC in the spleens of mice injected with SRBC or CPS-K A kinetic study was made of the increase in the numbers of direct PFC, indirect PFC and RFC for SRBC in the spleens following injection of SRBC or CPS-K (Fig. 1). In the spleens of normal untreated mice, there were small numbers of background PFC and large numbers of background RFC. The numbers of both direct PFC and RFC were found to be increased by 3 days after i.p. injection of SRBC. The highest levels of direct PFC and RFC were reached 4 or 5 days after injection, and their levels decreased thereafter. The number of indirect PFC was found to be increased by 5 days after injection of SRBC,
232
2L Nakashima, T. Kojima & N. Kato 1000
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Figure 1. Increase in direct PFC, indirect PFC and RFC for SRBC in the spleens of mice injected with SRBC or CPS-K. Mice were injected i.p. with 2x 108 SRBC (x), i.m. with 2 x 108 SRBC (A) or i.m. with 1000 pg of CPS-K (o). Each point represents ) or total numbers/spleen (- - - -) (x 10-3) of six to twelve mice with the geometric mean of numbers per 106 spleen cells ( its standard deviation.
the highest level was reached at 10 days, and its level decreased thereafter. After i.m. injection of SRBC, the number of direct PFC, indirect PFC and RFC were increased in similar fashions to after i.p. injection, although their peak levels were much lower. On the other hand, the number of direct PFC increased more rapidly after i.m. injection of CPS-K than after i.m. or i.p. injection of SRBC. The peak level of direct PFC after i.m. injection of CPS-K was much higher than that after i.m. injection of SRBC, although it was lower than the peak level after i.p. injection of SRBC. The number of direct PFC in the spleens of mice injected with CPS-K decreased almost as rapidly as those in the spleens of mice injected with SRBC. Despite such a marked increase in the numbers of direct PFC, the numbers of indirect PFC and RFC were not increased significantly after injection of CPS-K. The peak level of direct PFC in the spleens of mice injected with CPS-K was approximately the same as the level of RFC in the same mice. Relationship between direct PFC and RFC populations in the spleens of mice injected with CPS-K From the experimental results that in the spleens of mice injected with CPS-K the number of direct PFC at the peak approximated the number of RFC, a question arose as to whether direct PFC generated after CPS-K injection and RFC belonged to distinct or the same cell populations. The positions of the individual PFC and RFC, which were developed in
the same Cunningham's chamber, were recorded before and after incubation of the chamber (Table 1). Some RFC disappeared after incubation for 1 h at 370, whereas small numbers of RFC appeared at the new positions after incubation. These phenomena may be related to the appearance and disappearance of receptors for SRBC on the cell surface of lymphocytes (Warner, 1974) during incubation. On the other hand, only small numbers of direct PFC appeared at the position where an RFC was recorded before incubation. Most of the direct PFC appeared at the position unrelated to the position of RFC. These results indicate that most, if not all, of the RFC and direct PFC are distinct cell populations. This finding agrees with that of Wilson (1971) that PFC in the spleens of mice immunized with SRBC had no antigen-binding receptors demonstrable at 4°. Increase in the number of PFC for various kinds of erythrocytes in the spleens of mice injected with CPS-K and its correlation with increase in the total number of spleen cells In the experiments described so far, PFC and RFC for SRBC were examined. A study was then made to see whether PFC and RFC for other kinds of erythrocytes would also be increased by injection of CPS-K (Table 2). There were different numbers of background PFC and RFC for SRBC, RRBC and CRBC in normal untreated mice. The number of background RFC for CRBC was more than that for SRBC, whereas the number of background RFC for
Antibody synthesis stimulation by CPS-K
233
Table 1. Distinction between populations of PFC and RFC in the spleens of mice injected with CPS-K*
RFC numbers/chamber
Experiment number
Time of examination
I
Before incubation After incubation
2
32 13 At the positions where RFC was detected before incubation 9 21 12 At the positions where RFC was detected before incubation 11
Before incubation After incubation
PFC numbers/chamber 0 14
At the positions where RFC was detected before incubation 2 0 2 At the positions where RFC was detected before incubation 0
At new positions 4
At new positions 1
At new positions 12 At new positions 2
* For details see Materials and Methods section. Spleens were obtained from mice 3 days after the direct injection of 100 pg of CPS-K into the spleen. Table 2. Increase in the numbers of PFC and RFC for various kinds of erythrocytes and the total numbers of cells in the spleen of mice injected with CPS-K
Experiment Materials* number injected
1
3
3
88
102 3
None CPS-K
58
53
None CPS-K
4
2
None CPS-K
Spleen cell numbers (x 10-6) 71
None CPS-K
2
Days after injection
136
63 4
120
PFC and RFC numbers
Target
PFC/spleen
PFC/106 cells
RFC/spleen
RFC/106 cells
< 50 < 50
270+ 730 1360+410 313+270 3400+ 200 < 50 < 50 360+ 90 2440+ 90 765+ 490 23,700+ 7700 < 50 760+ 580 765+ 405 5100+ 500 2970+ 810 49,000+ 9500 112+22 335+ 155
1*0 > 2-0 None
CPS-K
SRBC
115 720 350 68 6950 1920 6350 1300 1180 550 55 580
0 2
0 0
0 0
6 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
* For details see Materials and Methods section. t Mice were injected i.p. with 100 pg of CPS-K or 5x 107 SRBC 12 days after i.v. injection of foetal liver cells. PFC and haemolytic foci were determined 4 days after injection of CPS-K or SRBC.
Increase in the numbers of direct PFC and haemolytic foci for SRBC by injection of SRBC or CPS-K in the spleens of mice thymectomized, irradiated and reconstituted with foetal liver cells A study was then made to see if and how injection of SRBC or CPS-K would increase the numbers of direct PFC and haemolytic foci in the absence of T cells (Table 6). There were considerable numbers of background PFC in the spleens of mice which were thymectomized, irradiated and reconstituted with foetal liver cells. Injection of SRBC into these mice increased only slightly the number of direct PFC. In contrast, much larger numbers of direct PFC were generated after injection of CPS-K. No large haemolytic foci appeared in the spleens of these mice, although some small haemolytic foci appeared in the spleens of some control mice and mice injected with CPS-K. These results indicate that in the absence of T cells CPS-K directly affects AFCP (B cells), whereas SRBC cannot do this. Moreover, it is suggested that the helper action of T cells is essential for the development of haemolytic foci.
Dependence on cell division of the increase in the numbers of direct PFC and RFC in the spleens of mice injected with CPS-K or SRBC To see if and how the increase in the numbers of
direct PFC and RFC in the spleen by injection of CPS-K or SRBC depends upon cell division, the sensitivities to vinblastine sulphate of total spleen cells, PFC and RFC in mice injected with them and in untreated control mice were determined (Table 7). The sensitivities of these cells to the drug are expressed as the ratios of the values for mice treated with the drug and for mice not treated. A single injection of vinblastine sulphate into normal control mice decreased the numbers of total spleen cells and background PFC and RFC for SRBC. The degree of decrease for background PFC was lower than that for total spleen cells, whereas the degree of decrease for background RFC was similar to that for total spleen cells. These effects of vinblastine sulphate on the normal spleens are perhaps related to its nonspecific cytotoxic action rather than to its mitosis inhibitory action. Because the total number of spleen cells was changed by administration of vinblastine sulphate, the sensitivities to the drug of PFC and RFC for different groups of mice should be compared by use of the values per 106 spleen cells. From these results, increase in the numbers of direct PFC and RFC by injection of SRBC appeared to be markedly dependent on cell divisions. Increase in the number of direct PFC by injection of CPS-K appeared to be definitely dependent on cell divisions. The number of RFC was increased only very slightly by
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injection of CPS-K, but cell divisions appeared to be required to some extent for maintenance of RFC in these mice. Increase in the total number of spleen cells appeared also to be definitely dependent on cell divisions, suggesting that a large part of total spleen cells are affected by mitogenic action of CPS-K. The dependency on cell divisions of development of PFC in these mice appeared to be slightly higher than that of increase in total spleen cells. On the other hand, the dependency on cell divisions of development of RFC appeared to be lower than that of increase in total spleen cells. These results indicate that cell divisions are required for the increase in the number of direct PFC and for maintenance of RFC in mice injected with CPS-K, and that CPS-K induces proliferation and differentiation of different subpopulations of spleen cells to different degrees. DISCUSSION In the spleens of mice, IgM AFC for SRBC antigen are generated by the non-specific stimulus (CPS-K) almost as markedly as by the specific antigenic stimulus (SRBC). With the use of this experimental system, we compared the modes of increase in IgM AFC and IgG AFC and their precursors by the
specific and non-specific stimuli. Evidence has been presented to support the idea that increase in the number of IgM AFC (direct PFC) in the spleen of mice injected with CPS-K does not result from very frequent divisions of individual AFC or their precursors. First, injection of CPS-K did not increase the number of haemolytic foci (Table 5), which would appear through clonal proliferation of AFC or their precursors (Playfair et al., 1965). It may therefore be that injection of CPS-K generates direct PFC diffusely from relatively large numbers of the precursor cells through a few or no cell divisions, although another possibility remains to be excluded that dividing cells stimulated by SRBC stay together in one place but cells stimulated by CPS-K do not. Secondly, the number of total spleen cells was not increased greatly by injection of CPS-K, while the number of direct PFC for various antigens was increased markedly (Table 2). It appears that CPS-K, like other B-cell mitogens (Coutinho and Moller, 1975), stimulates B cells, which can be regarded as AFCP for numerous antigens and occupy approximately 50 per cent of total spleen cells (Greaves et al., 1973), non-speci-
fically to generate AFC without the help of action of T cells (Table 6). It has not been estimated what percentages of B cells in the spleens are stimulated by CPS-K. It may be reasonable, however, to consider that the total spleen cells would have increased more markedly if considerable numbers of B cells divide very frequently. Thirdly, we found that injection of CPS-K increased the number of RFC only very slightly (Fig. 1, Tables 2 and 4). It appears that most of RFC which we estimated in the present study are B cells and therefore AFCP, although the possibility that a small proportion of them may be T cells, macrophages and AFC themselves could not be completely ruled out (see Materials and Methods section and Table 1). This may therefore indicate that CPS-K does not induce marked proliferation of AFCP (B cells). This lack of stimulatory effect of CPS-K on the proliferation of RFC is possibly related to the fact that increase in immunological memory is not induced in mice iniected with CPS-K (Table 4) It has been recently shown that AFC can be generated from their precursors without cell divisions (Marbrook and Haskill, 1974; Sulitzeanu et al., 1973; Andersson and Melchers, 1974), while other investigators showed that at least one cell division is required for the generation of AFC from their precursors (Nakamura, Segal, Globerson and Feldman, 1972). In the present study, it has been shown that cell division is probably required for the generation of AFC from the precursors by CPS-K (Table 7). In mice injected with CPS-K direct PFC are increased to as high levels as background RFC (Fig. 1 and Table 2). A possible mechanism of the generation of AFC by the non-specific stimulus of CPS-K is that a large number of background RFC are stimulated by CPS-K to differentiate to direct PFC through one cell division or a few divisions. CPS-K causes no definite changes in RFC levels, but cell division is required to some extent for the maintenance of RFC at the same level as background RFC (Table 7). It may therefore be that RFC are replaced by newly differentiating precursors but the rate of generation of new RFC do not exceed that of differentiation of RFC to PFC. The alternative possibility may exist, however, that RFC respond to SRBC and a T-cell factor only and direct PFC stimulated by CPS-K come from the other cell population than RFC. Only very small numbers of IgG AFC (indirect PFC) were generated after a single injection of CPS-K, even after repeated injections (Fig. 1 and
Antibody synthesis stimulation by CPS-K Table 4). It has been reported that there are only small numbers of precursor cells for IgG AFC in untreated mice (Warner, 1974). For the marked increase in IgG PFC, therefore, the precursor cells may have to divide very frequently. Alternatively, if the switch over from IgM AFC (or their precursors) to IgG AFC (or their precursors) is the case, it may be accomplished only through frequent cell divisions of the precursor cells. In any case, the lack of frequent cell divisions of AFCP in the spleens of mice injected with CPS-K may be the cause of the fact that IgG PFC are not increased by the non-specific stimulus of CPS-K. A hypothetical model which would summarize the results in this work is shown in Fig. 2. It may be that the stem cells (or immature B cells) generate RFC (there may also be stepwise functional maturation in subpopulations of RFC) and some of the mature RFC differentiate to direct PFC in the microenvironR
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