Immunology 1979 38 569

Anti-DNP antibody responses to DNP-histone Hi in mice

S. ISHIZAKA, S. OTANI & S. MORISAWA Department of Biochemistry, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka, Japan

Acceptedfor publication 14 June 1979

classes of histone which are designated as HI, H2A, H2B, H3 and H4. Amino acid sequence determinations have revealed that the arginine-rich histones H3 and H4 are rigidly conserved and histones H2A and H2B are also conserved to a considerable extent, while the very lysine-rich histone H I is the least conserved of all histones (Rall & Cole, 1971; Macleod, Wong & Dixon, 1977). Although the immunogenicity of these histone fractions is relatively weak because of the similarity in their structures between different species, many attempts have been made to prepare specific antibodies to each histone fraction to study the exact organization of chromosomal components which may undergo temporal variations during the life cycle of a cell (Bustin, 1976). These antisera are also useful in comparative studies of histone fractions of different species (Stollar & Ward, 1970; Bustin & Stollar, 1972; Bustin & Stollar, 1973a; Sluyser & Bustin, 1974). In addition, anti-histone antibodies are frequently detected in the sera of patients with autoimmune diseases such as systemic lupus erythematosis (Holman & Kunkel, 1957; Holman, Deicher & Kunkel, 1959). Less information is available, however, concerning how antibody responses to histone components are induced, although antigenic determinants have been carefully analysed, especially in histone HI (Bustin & Stollar, 1973b). We have attempted to investigate antiDNP antibody responses to DNP-histone H I in mice. This is because free histone fractions are very weak immunogens and because histone HI differs from other histone fractions by exhibiting a higher multiplicity and a certain degree of species specificity (Stollar

Summary. Anti-DNP antibody responses to DNP-histone HI in C3H/HeN mice were not suppressed by in vivo treatment with carrageenan in which many phagocytic macrophages were presumed to be impaired. Rather higher antibody responses to this antigen were observed in athymic nude mice than in heterozygous nude mice. Further, non-adherent spleen-cell and T-cell depleted spleen cells induced in vitro anti-DNP antibody responses to DNP-histone HI to the same extent as normal spleen cells. These results suggest that anti-DNP antibody responses to DNP-histone HI are macrophage- and thymus-independent. It was also observed that IgG-type anti-DNP antibodies to DNPhistone HI were produced although most thymusindependent antigens were shown to induce predominantly IgM type antibodies but little, if any, IgG type antibodies. Furthermore, histone HI did not show any polyclonal B-cell activator activities in contrast to many other thymus-independent antigens which act as polyclonal B-cell activators. INTRODUCTION

The histones are basic proteins associated with DNA in the somatic cells of all eukaryotes but not in prokaryotes. In most mammalian tissues, there are five Correspondence: Dr Seiji Morisawa, Department of Biochemistry, Osaka City University Medical School, 1-4-54 Asaki-machi, Abeno-ku, Osaka 545, Japan. 0019-2805/79/1 100-0569$02.00 © 1979 Blackwell Scientific Publications

569

570

S. Ishizaka, S. Otani & S. Morisawa

& Ward, 1970; Bustin & Stollar, 1972; Bustin & Stollar, 1973 a, b; Sluyser & Bustin, 1974).

were estimated to be 0-63 + 0.07%; otherwise 424+0-41% phagocytes were contained in normal spleen cell suspensions.

MATERIALS AND METHODS

In vitro antibody responses to DNP-histone HI Cultures of 2 x I07 cells/ml of spleen cells, non-aherent spleen cells or T-cell depleted spleen cells from unprimed or immunized C3H mice were prepared according to the method of Marbrook (1967). As the medium of cultivation, RPMI 1640 medium (Nissui Seiyaku Co.) was used with the addition of IO% horse serum and 60 pg/ml of kanamycin. These cells were stimulated with various amounts of DNP-H 1, 1 pg/ml of DNP-BGG or 3 x 106 of SRBC and cultured in a humidified cell incubator at 38° for 4 days with a constant gas flow (5% CO2 in air).

Animals C3H/HeN crj, BALB/cA nu/nu and BALB/cA nu/+ male mice, all 8-10 weeks old, were used in these experiments. C3H/HeN crj mice were purchased from Charles River Co., Japan. Homozygous BALB/cA nude mice and heterozygous nude mice (BALB/cA nu/ +) were obtained from CLEA Japan INC. Immunogens

DNPO.8-histone HI (DNP-H 1), DNP4-bovine serum y-globulin (DNP-BGG), DNP17-ovalbumin (DNPOVA), sheep red blood cells (SRBC) and TNP4-lipopolysaccharide (TNP-LPS) were used as the antigens. DNP-H 1, DNP-BGG and DNP-OVA were prepared according to the method of Eisen, Carsten & Belman (1954). TNP-LPS was prepared by the method of Rittenberg & Pratt (1969) with a slight modification. The number of DNP or TNP groups per molecule of conjugated materials was calculated as described by Eisen et al. (1954) and Rittenberg & Ankraut (1966), respectively. The approximate molecular weights of histone HI (from calf thymus, Sigma Chemical Co.), BGG (Sigma Chemical Co.), OVA (Grade V, Sigma Chemical Co.) and LPS (from E. coli 01 11 :B4, Difco Laboratories) were estimated to be 2 1 x 104, 4 5 x 104 and 5 x 105 respectively.

Removal of adherent cells from spleen cells Spleen cell suspensions (5 x 107 cells/ml) were prepared in RPMI 1640 medium supplemented with 10% horse serum and incubated in plastic petri dishes at 370 for 1 h as described previously (Ishizaka et al, 1977). Non-adherent cells were collected by washing the dish with the same medium followed by transfer to another dish. Further incubation was performed for 30 min under the same conditions. Finally, the non-adherent spleen cells were harvested and adherent cell-depleted cell suspensions containing 2 x 107 cells/ml were prepared. It was shown that these cell suspensions contain 0-0 1 9% phagocytes by the uptake of latex particles.

Immunization Various doses of DNP-H 1, 100 jug of DNP-BGG and 100 pg of DNP-OVA were emulsified in Freund's complete adjuvant and injected into the peritoneal cavity of each mouse. In the case of TNP-LPS, antigen (1 pg) was injected in the same manner without adjuvant.

Removal of T cells from spleen cells T-cell depleted spleen cell suspensions were prepared by treatment of spleen cell suspensions from normal or DNP-HI primed C3H/HeN mice with anti-0 serum and complement (Gorczynski, Miller & Phillips, 1972). Anti-O serum was prepared by immunizing AKR/J mice with thymus cells obtained from C3H/HeN crj donors (Gorczynski et al., 1972).

Treatment of mice with carrageenan Carrageenan (potassium salt, from Irish moss, Sigma Chemical Co.) was injected into each mouse intraperitoneally as described previously (Ishizaka, Otani & Morisawa, 1977). Carrageenan (0 5 mg) dissolved in physiological saline was injected intraperitoneally into each mouse every 2 days for 1 week and the antigen was given one day after the last injection of carrageenan. The phagocytes contained in the spleen cell suspension I day after the last injection of carrageenan

Assay of antibody-forming cells Trinitrophenyl group-conjugated sheep red blood cells (TNP-SRBC) were prepared according to the method of Rittenberg & Pratt (1969) and Harrell & Merchant (1967) respectively for determination of the number of plaque-forming cells. The numbers of antibody-forming cells to hapten, SRBC and chicken red blood cells (CRBC) were estimated by Jeme's haemolytic plaque assay (Jerne & Nordin, 1963). Indirect anti-DNP PFC were determined by the method of

571

Anti-DNP antibody responses Dresser and Wortis (1965) with rabbit anti-mouse IgG serum (Miles Laboratories Inc.)

RESULTS Primary anti-DNP antibody

responses

estimated to be 0 63 + 0.07%, 0 33 + 0.09% and 025 + 0.19% 1 day, 3 days and 7 days after carrageenan treatment respectively, while 4 20 + 0.38% phagocytes were contained in normal spleen cell sus-

were

pensions.

to DNP-H1 in

vivo

Effects of carrageenan treatment on anti-DNP antibody responses to DNP-H 1 in C3H/HeN mice were investigated to determine whether the in vivo antibody responses were macrophage-dependent. As shown in Table 1, anti-DNP antibody responses to DNP-H 1 ranging 10-2 to 100 pg were not suppressed by carrageenan when tested 3 days after immunization. The production of anti-DNP antibody to DNP-BGG, however, was significantly reduced by carrageenan treatment. Furthermore, it was noted that the antiDNP antibody responses of athymic nude mice to DNP-H1 were higher than those ofheterozygous nude mice, while anti-DNP responses to DNP-OVA, a thymus- and macrophage-dependent antigen, were higher in heterozygous mice than in athymic nude mice (Table 1). The percentages of phagocytes in spleen-cell suspensions were determined by the inclusion test of latex particles after carrageenan treatment to confirm the cytotoxic effects of carrageenan on phagocytes. The phagocytes contained in spleen-cell suspensions

Kinetics of primary anti-DNP antibody responses to DNP-Hl and TNP-LPS When DNP-H1 (10 ug) emulsified in Freund's complete adjuvant or TNP-LPS (1 jg) was injected to carregeenan-treated or untreated C3H/HeN mice intraperitoneally and anti-hapten PFC were determined, it was shown that anti-DNP antibody responses to DNP-HI were not suppressed significantly by carrageenan for at least 7 days. On the other hand, antiTNP antibody responses to TNP-LPS were reduced significantly by carrageenan treatment as described in a previous report (Ishizaka et al., 1977). These results are shown in Fig. 1.

Primary IgM anti-DNP responses to DNP-H1 in vitro Normal spleen cells, non-adherent spleen cells or T-cell depleted spleen cells prepared from C3H mice were cultured with various concentrations ofDNP-H I for 4 days and IgM anti-DNP PFC were estimated. Non-adherent spleen cells and T-cell depleted spleen

Table 1. Anti-DNP responses to DNP-Hl in vivo. Various doses of DNP-Hl and 100 pg of DNP-BGG both emulsified in Freund's complete adjuvant were injected to the carrageenantreated or untreated C3H/He mice. The numbers ofPFC per spleen were estimated 3 days after the immunization with DNP-H 1

Strains

C3H/He

Antigens

Carrageenan

(pg)

treatment

DNP-H1

10-2 10-2

DNP-HI DNP-HI

1 1

DNP-HI

DNP-H1 DNP-H1 DNP-H1 DNP-H1 DNP-BGG BALB/cA nu/+ BALB/cA nu/nu BALB/cA nu/+ BALB/cA nu/nu

Dose

DNP-BGG DNP-HI DNP-HI DNP-OVA DNP-OVA

10 10 100 100 100 100 10 10 100 100

_ +

+ + + + -

Anti-DNP PFC/spleen + SE

Student's t test

6,840+561 11,200+2061 10,980+ 1250 16,533 + 2226 11,600+ 367 18,667+ 1653 10,170+ 1563 14,900+2333 33,025 + 891 22,275 + 702 3,967+668 7,033 + 707 14,000+ 1500 6,750+ 614

0 1> P 0 5> P> 001

0I>P>o001

* The number of PFC was estimated 7 days after immunization with DNP-BGG or DNP-OVA. All values are means and standard errors of triplicated experiments.

572

S. Ishizaka, S. Otani & S. Morisawa DNP-H 1 (10 pg) were prepared I month after immunization, and they were cultured in the presence of various amounts of DNP-H1 ranging from 10-4 pg/ml to 10 pg/ml. When IgG anti-DNP PFC were assayed after 4 days of cultivation by indirect haemolytic plaque assay (Dresser & Wortis, 1965), it was shown that anti-DNP responses to DNP-H1 in non-adherent spleen cells or T-cell depleted spleen cells were not lower than those in spleen cells as shown in Table 3. The removal of adherent cells or T cells, however, caused a marked suppression of anti-DNP responses to DNP-BGG, a typical thymus- and macrophagedependent antigen (Table 3).

U_~~~ Q

0

0

0

WI

3 5 7 Days after immunization

Figure 1. Kinetics of antihapten antibody responses to DNP-H1 and TNP-LPS in vivo. C3H mice were immunized with 10 pg of DNP-HI emulsified in Freund's complete adjuvant or 1 ,ug of TNP-LPS. The number of PFC was assayed after the indicated days. o, Number of anti-DNP PFC to DNP-H1 in spleen cells; *, number of anti-DNP PFC to DNP-H I in spleen cells from the carrageenan treated mice; &,

number of anti-TNP PFC to TNP-LPS in spleen cells;

Histone Hl is not a polyclonal B-cell activator Since it is well known that many thymus-independent antigens induce polyclonal antibody production, experiments were designed to study whether histone H1 has the same action on antibody responses using SRBC, CRBC, TNP-SRBC and BPO-SRBC as antigens. When unprimed C3H mouse spleen cells (1 x 107 cells/ml) were cultured with various concentrations of histone Hl or 150 pg of LPS in serum-free medium, and anti-SRBC, anti-CRBC or anti-hapten PFC were determined, histone H1 did not show any evidence of polyclonal B-cell activation, while typical polyclonal antibody responses were observed with LPS. These results are shown in Table 4.

A,

number of anti-TNP PFC to TNP-LPS in spleen cells from the carrageenan-treated mice. Each point and vertical bar gives the geometric mean and standard errors of triplicate experiments.

cells responded to DNP-H1 to the same extent as or somewhat more than normal spleen cells. The removal of adherent cells or T-cell depletion, however, caused a marked decrease in responses to SRBC as shown in Table 2. The results obtained in non-adherent spleen cells were consistent with the results of in vivo experiments in which phagocytes were impaired by carrageenan treatment. The combined experimental results suggest that anti-DNP antibody responses to DNP-H 1 may be macrophage-independent as well as T-cell independent.

Secondary IgG anti-DNP responses to DNP-H1 in vitro Spleen cells, non-adherent spleen cells or T-cell depleted spleen cells from C3H mice pre-immunized with

DISCUSSION The number, type and chemical structure of most histones found in eukaryotic cells differ little from one organism to another (Delange & Smith, 1971). Therefore, it is expected that antigenicity of histones may be weak compared with that of other proteins. Among histone fractions, lysine-rich histone (H1) exhibits a higher multiplicity and a certain degree of species specificity. This is one reason why we chose dinitrophenylated histone H 1 as an antigen in our experiments. Anti-DNP antibody responses of C3H mice to dinitrophenylated calf thymus histone H 1 (DNP-H 1) were, however, rather weak. This is also the case in other mouse strains such as AKR, C57B1/6, BlOD2 and B1O (data not shown). In the present report, we suggest that anti-DNP antibody responses to DNP-H 1 in mice are macrophage-independent as well as thymus-independent, because the antibody responses are not sup-

Anti-DNP antibody responses

573

Table 2. Primary anti-DNP responses to DNP-HI in vitro. Spleen cells (2 x 107/ml), non-adherent spleen cells (2 x 107/ml) or T-cell depleted spleen cells (2 x 107/ml) from the unprimed C3H/He mice were cultured with various concentrations of DNP-H1 or 3 x 106 SRBC/ml for 4 days as described in Materials and Methods. IgM type anti-DNP PFC or anti-SRBC PFC per culture were assayed Cells

Antigens

Spleen Nonadherent*

DNP-HI 10-4pg

128+53

DNP-HI 10-4 ug

300 + 58 187+36 220+ 56 355 + 26 406+85 225+47 370 + 62 517+79 235 + 56 377 + 38 295 + 67 1600+270 170+ 70 320+42

Bt Spleen Non-adherent B Spleen Non-adherent B Spleen Non-adherent B Spleen Non-adherent B

Anti-IgM PFC/culture ± SE

Dose

DNP-H1 10-4ug DNP-HI 10-2 lg

DNP-HI DNP-HI DNP-HI DNP-HI DNP-H1 DNP-H1 DNP-HI DNP-HI SRBC SRBC SRBC

10-2 Mg 10-2 lg

I Mg 1 pg lg 10ug 10 Mg 10jig 3 x 106cells 3 x 106 cells 3x

106cells

* Non-adherent spleen cells were prepared from the unprimed C3H mice as shown in Materials and Methods. t T-cell depleted spleen cells were prepared as described in Materials and Methods. All numbers represent the values of PFC after back ground PFC reduction. Anti-DNP PFC background of the spleen cells, non-adherent spleen cells and T-cell depleted spleen cells were 39 + 2, 41 + 3 and 54 + I 1, respectively. All values are means and standard errors of triplicated experiments.

Table 3. Secondary anti-DNP responses to DNP-H I in vitro. Spleen cells (2 x 107/ml) were prepared from the mice pre-immunized with DNP-H1 (10 pg) one month previously. They were cultured with various amounts of DNP-H1 or I pg/ml of DNP-BGG for 4 days and the indirect PFC per culture were estimated. All values represent the means and standard errors of tripilicate experiments

Antigens

IgG anti-DNP PFC/culture + SE Dose (pg/ml) Spleen cells Non-adherent cells T depleted cells 0

DNP-HI DNP-H1 DNP-H1 DNP-H1 DNP-BGG

10-4 10-2 1 10 1

165+5 217+39 203+29 617+59 190+47 1300+ 69

167+15 370+21 415+75 675+25 483 + 78 325 + 17

390+33 760+34 893+99 1367+ 135 483 +42 265 + 54

S. Ishizaka, S. Otani & S. Morisawa

574

Table 4. Effects of HI on the induction of polyclonal antibody responses. Spleen cells (107 cells/ml) from the unprimed C3H mice were cultured with various concentrations of H 1 or 150 ig of LPS in serumfree medium. The number of direct PFC per culture was measured after 2 days. All experiments were performed in triplicate

Antigens

Histone l

LPS

Dose Anti-SRBC Anti-CRBC Anti-TNP Anti-BPO (pg) PFC + SE PFC + SE PFC + SE PFC + SE 0 10 100 500 1000 150

10+3 6+2 5+2 5+2 0 135+15

pressed by in vivo carrageenan treatment of mice (Table 1), and either non-adherent spleen cells or T-cell depleted spleen cells produce anti-DNP antibody in vitro to the same extent as untreated spleen cells (Table 2). The fact that the antibody responses to DNP-H 1 are somewhat higher in athymic nude mice than in heterozygous nude mice may also support this explanation. It was suggested, however, that thymusindependent antigens may only require a very small number of macrophages but are still macrophagedependent (Lee, Shiozawa, Shaw & Diener, 1976). In this regard, it should be explained that anti-DNP antibody responses to DNP-H 1 are relatively macrophage-independent because macrophages are not removed completely under our experimental conditions. We should also add the apparent requirement for macrophages in anti-TNP responses to TNP-LPS (Fig. 1), although responses to LPS or its conjugates have been described as both macrophage-dependent and independent (Coutinho, 1975; Kagnoff, Billingo & Cohn, 1974). Most thymus-independent antigens induce predominantly IgM-type antibodies but little, if any, IgGtype antibodies. The latter, however, were also produced in our experiments in vitro (Table 3). This does not appear to be a special case in DNP-H 1 since potent B antigens such as DNP-Ficoll can induce some IgG antibody synthesis (Mosier, Johnson, Paul & McMaster, 1974). Furthermore, it was proposed that many thymus-independent antigens act as polyclonal B-cell activators resulting in polyclonal antibody responses (Coutinho & Moller, 1973). This is the case in antiDNP responses to DNP-casein and DNP-gelatin as

43+5 25+11 15+3 15+3 NT 85+15

104+41 70+16 43+6 68+ 14 60+26 1435+150

13+9

3+1 2+ 1 8+3 5+2 73+13

reported previously (Ishizaka, Otani & Morisawa, 1979). As shown in Table 4, however, the mitogenicity of histone H 1 could not be demonstrated in our experiments which were performed using various concentrations of histone H1. We used DNP-H I with a low epitope density because it was suggested that compounds of low epitope density were exclusively immunogenic, while those heavily conjugated were solely tolerogenic (Desaymond & Feldmann, 1975). It remains to be determined whether antigens with higher epitope densities elicit the same profiles of antibody responses. Studies on the molecular nature of thymus-independent antigens revealed that a polymer structure with repeating antigenic determinants and slow metabolism are responsible for T-cell independency. DNP-histone H 1 may not have such characteristics, while it was suggested that the histones increase the phagocytic activity of macrophages (Agbaba, 1976). We feel that there are many problems to be solved before the general properties of thymus-independent antigens and their triggering mechanisms of antibody responses are conclusively determined.

ACKNOWLEDGMENTS We wish to thank Miss M. Ishimoto for her skilled technical assistance. This study was supported by a grant from the Ministry of Education of Japan.

Anti-DNP antibody responses

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Anti-DNP antibody responses to DNP-histone H1 in mice.

Immunology 1979 38 569 Anti-DNP antibody responses to DNP-histone Hi in mice S. ISHIZAKA, S. OTANI & S. MORISAWA Department of Biochemistry, Osaka C...
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