Immunology 1978 34 363
Delayed hypersensitivity in mice induced by intravenous sensitization with sheep erythrocytes: evidence for tuberculin type delayed hypersensitivity of the reaction A. MITSUOKA, T. TERAMATSU, M. BABA,*t S. MORIKAWA*t & K. YASUHIRA* Department of Thoracic Surgery and *Department of Pathology, Chest Disease Research Institute, Kyoto University, Kyoto 606, Japan
Received 10 January 1977; acceptedfor publication 8 August 1977
Summary. Delayed hypersensitivity (DH) reaction
cutaneous sensitization with methylated human serum albumin (MHSA) in FCA. Taken collectively, it was concluded that this DH reaction can be categorized as the tuberculin type.
can be induced in mice by intravenous sensitization with sheep erythrocytes (SRBC). However, as the
sensitizing procedure is quite different from a usual mode of sensitization for DH using complete Freund's adjuvant (FCA), the nature of this reaction
INTRODUCTION
has been a matter of controversy. In an attempt to characterize this reaction, we placed special interest on two possibilities regarding the nature of this reaction; Jones-Mote reaction or tuberculin type DH. From the kinetics study on the DH after challenge, the DH reaction to SRBC in mice by intravenous sensitization was clearly distinguished from the Arthus reaction. The dose-response pattern of this reaction also suggested that the contribution of Arthus reactivity to delayed reactivity was negligible. Cell reconstitution experiments revealed this DH to be quantitatively thymus cell dependent. Furthermore, this DH required macrophages at its manifestation stage, and appearance of basophil infiltration at the lesion was absent. In addition, strain difference and ageing of host mice influenced the DH reaction in exactly the same fashion in which these factors influence the tuberculin type-DH induced by sub-
Delayed hypersensitivity (DH) to sheep erythrocytes (SRBC) in mice was reported for the first time by Nelson & Mildenhall (1967), who used a subcutaneous or intraperitoneal sensitization and the footpad test. According to their observations, sensitization of mice with SRBC could induce a strong DH reaction when Freund's complete adjuvant (FCA) was the sensitizing carrier, and slight DH reaction when Freund's incomplete adjuvant (IFA) or saline was the carrier. The DH to SRBC induced without FCA was considered to be a Jones-Mote reaction as described by Raffel & Newel (1958), and tuberculin-type DH was considered to develop only by sensitization with FCA when the antigens were soluble proteins or heterologous erythrocytes as reviewed by Crowle (1975). Recently, the development of DH in mice to SRBC was again observed by Miller, Mackaness & Lagrange (1973), who used a subcutaneous sensitization with SRBC in saline. Intraperitoneal sensitization with SRBC in saline also appeared to produce a considerable skin DH in mice (Kerckhaert, 1974). One of the most interesting findings was
t Present address: Department of Pathology, Shimane Medical School, Izumo 693, Japan. Correspondence: Dr Akio Mitsuoka, Department of Thoracic Surgery, Chest Disease Research Institute, Kyoto University, Kyoto 606, Japan.
363
364
A. Mitsuoka et al.
the demonstration of the ability of mice to develop DH after intravenous sensitization with SRBC in saline (Kettman, 1972). These reactions have been currently used in studies of the immune regulation involved in DH. However, based on the rapid kinetics of the reactions, these phenomena may also be considered a Jones-Mote reaction different from the tuberculin-type DH. In this report, the nature of the DH in mice, as induced by intravenous sensitization with SRBC, was investigated in an attempt to clarify whether such belongs to the tuberculin-type of Jones-Mote type DH.
MATERIALS AND METHODS Mice AKR/J BALB/c Crgl, and C3H/HeMs mice bred and fed in the animal house of CDRI, Kyoto University, and C57BL/6 and BDF1 mice from the Experimental Animal Cooperative Association of Shizuoka (Hamamatsu) were used. Detailed records included age and sex of mice are described in the text or the footnotes.
Antigen SRBC were obtained in Alsever's solution from the Nikken Animal Blood Supply Centre (Kyoto). These cells were stored at 0-4' for less than 30 days and were washed three times and suspended in phosphate buffered saline (PBS, pH 7-1) before use. Sensitization
Mice were sensitized by an intravenous administration into the retro-ocular plexus of a solution of 0-2 ml consisting of varying doses of SRBC. Assay for skin reaction A challenge injection of about 108 SRBC in 0-02 ml of PBS was given into the right footpad at various intervals after sensitization. As a standard schedule for DH, the interval of 4 days was adopted for reasons described later. The method for measurement of the footpad reaction (FPR) was fully described in our previous paper (Baba, Harada & Morikawa, 1977). In most experiments, thickness of both the right and left foot was measured at 24 h after challenge just following exanguination, and the difference between these two measurements was described in 1/10 mm (FPR) as the intensity of
DH. In some experiments, FPR was measured at different intervals after challenge other than 24 h.
Assay for antibody producing cells The number of IgM-plaque forming cells (PFC) against SRBC of the spleen was assayed by the method of Cunningham & Szenberg (1968). Titration of serum antibody Peripheral blood was obtained just before measurement of FPR. Serum was separated from the blood, pooled from each group of mice and stored at - 1520° until assay. Titration of haemagglutinin (HA) and haemolysin (HL) of the serum was performed using microtitration trays.
Histological methods After the intensity of FPR was measured, the footpad was excised and fixed in 10% formalin solution. The paraffin section was stained with haematoxylin and eosin. For the detection of possible infiltration of basophils, tissues were fixed in 5 % phosphate buffered glutaraldehyde (pH 7 3), postfixed in osmium tetroxide, and 1 pm epon embedded section were prepared. These sections were stained for 1 h in Giemsa solution diluted 1:10 in 2% sodium borate solution after the method by Dvorak, Dvorak, Simpson, Richerson, Leskowitz & Karnovski (1970). Alternatively, tissues were fixed in 10% formalin solution containing 1 % cetylpyridium chloride, the procedure of which can insolubilize heparin-containing metachromatic granules of basophils or mast cells. The fixed tissues were embedded in paraffin and the sections were stained with toluidine blue. Pretreatments of mice In one experiment, mice were thymectomized at the age of 2 months, and given a total body irradiation of 800 rad 12 days later. These mice were then reconstituted with the proper number of lymphoid cells on the same day of irradiation. The syngeneic donor mice were 4 weeks of age for thymus cell transfer, or 8 months for bone marrow cell transfer. The recipient mice were all checked after FPR measurement to confirm that the thymus had been totally excised. In another experiment, mice were injected intraperitoneally with 0-2 ml of 0-1 % ferritin solution. The ferritin solution was made by resolving
six times-crystallized ferritin from horse spleen (Pentex Biochemicals, Kankakee, U.S.A.) in PBS.
365
Delayed hypersensitivity to sheep erythrocytes RESULTS
Time effect on the development of DH to SRBC after sensitization Male C57BL mice aged 2 or 3 months were sensitized intravenously with 106 SRBC and given a challenge injection at varying intervals thereafter. FPR at 24 h after challenge was measured (Fig. 1). The optimal interval between sensitization and challenge was 4 days in group of either age. In the case of higher dose sensitization, the peak response was seen also on the fourth day, but the response was less intense (data not in figures).
14
12 I I
I I
10
I
I I
F
I
I
6
-
k
I
I
c-JIT
I
8
I1
I
I
I I I
Kinetics of FPR to SRBC after challenge Kinetics of FPR after challenge were investigated in mice sensitized with 106 SRBC intravenously at 2 months old (Fig. 2) or 3 months old (Fig. 3). In Fig. 2, a slight response was observed at 3 h and a prominent peak was seen at 24 h on the fourth day of challenge. When the mice were challenged 6 or 8 days after sensitization, a higher peak response at 3 h and a lower response at 24 h were seen. The peak response was seen to be delayed more than 24 h on the third dayofchallenge. TheFPRat 58h differed among each group, despite a similar serum HA titre. Similarly in Fig. 3, a marked peak response was observed at 24 h in the fourth day group. It is noted that the DH reaction in this group became apparent after 12 h and was still considerable at 48 h. A plateau reaction which increased from 24 to 48 h and subsequently diminished was observed in the third day group. Of interest is the fact that the Arthus reaction was different in intensity as seen in Figs 2 and 3, whereas the DH intensity showed a quite reproducible pattern in both cases (see Fig. 1). From these results, DH is apparently not influenced by the Arthus reaction, particularly in mice challenged on the fourth day. Thus, in further studies, we adopted the interval of 4 days for investigation of the DH to SRBC. Influence of sensitizing dose on DH and antibody production Male C57BL mice aged 3 5 months were sensitized with varying doses of SRBC and challenged 4 days later. FPR of each mouse was measured at 3 and 24 h after challenge. Serum HA and HL titres and splenic PFC were also assayed (Table 1).
I I -q Ln
6
I i
I
I
I \
4
T~~~~~~~~~~~~~4
-.I-
2
3
4
5 6 after sensitization Days
7
8
Figure 1. DH to SRBC in mice at various intervals after sensitization. Male C57BL mice aged 2 (0) or 3 (0) months were sensitized intravenously with 106 SRBC. FPR was measured at 24 h after challenge. Each group included five or more mice. Vertical bars are standard errors of the means.
A direct correlation was found between the sensitizing dose and Arthus reaction as well as PFC, HA and HL titres, particularly when the dose was over 106. On the other hand, as DH was the highest at the dose of 106, the presence of an optimal sensitizing dose in DH was confirmed.
Thymus dependency of DH to SRBC Male C57BL mice were thymectomized at the age of 2 months, 800 rad irradiated and reconstituted with lymphoid cells from normal syngeneic male mice. The recipient mice were then sensitized with 106 SRBC given intravenously followed by challenge 4
days thereafter.
366
A. Mitsuoka et al. IC'
E
6.0
5-5 63 U-~
~
~
17 ~
~
~
~
245 ~~~~~~~~~~~~-
2
-~~~~~~~~~~~~~~~~-
17
3 6
24
58
Hours after challenge
Figure 2. Kinetics of FPR to SRBC after challenge. Male C57BL mice aged 2 months were sensitized intravenously with 106 SRBC 3 (0-0), 4 (0-0), 6 (O - - 0) or 8 ( -- 0) days previously. Serum HA titre (log 2) at 58 h after challenge is also shown. Each group included five or more mice.
E8 6
4 0
;
3
/
12
24
48
72
Hours after challenge
Figure 3. Kinetics of FPR to SRBC after challenge. Male C57BL mice aged 3 months were sensitized intravenously with 106 SRBC 3 (0-0), 4 (0-0), 5 (O - - 0), or 6 ( -- 0) days previously. Each group included five or more mice.
Delayed hypersensitivity
to
sheep erythrocytes
367
Table 1. Delayed hypersensitivity and antibody production to varying doses of SRBC in mice
Antibody titret 3 h FPR*
Dose -
3-5+ 1-5 2-8+P19
05 106 107
3-8+2-7 4-2+ 1-7 5 0+ 2-4 6-2+ 13
lo8
109
24 h FPR* 3 0+2-0 7-6+ 1 7 15-4+3-2 10-7+ 1 9 3-4+ 1-6 1-2+ 10
PFC/spleent (x 100) 3-7
3-2 99 250 368 581
HA
HL
1.0 1.0 2-0 50 70 7-0
0 0 45 6-0 8-0 9-0
Male C57BL mice aged 3-5 months were sensitized intravenously with varying doses of SRBC, and challenged 4 days later. * Mean + standard error (1/10 mm). t Spleen cells and serum were obtained from mice at 24 h after challenge. Each group included five mice.
Table 2 shows that the intensity of DH increased in proportion to the number of thymus cells transferred together with a given number of bone marrow cells. Mice not given thymocytes did not develop detectable DH. Effect of fefritin on DH to SRBC In our previous work (Baba et al., 1977), ferritin was revealed to be as selectively toxic to macrophages as was carragenan. To determine the participation of macrophages in eliciting delayed footpad swelling, male BDF1 mice aged 6 weeks sensitized with 106 SRBC were given 0-2 mg of ferritin intraperitoneally just before footpad challenge on the
Table 2. T cell dependence of DH to SRBC in mice
fourth day of sensitization. As shown in Table 3, DH reaction was completely inhibited, while this treatment appeared to have no effect on the general condition of mice.
Histological studies on FPR to SRBC Histology of 24 h FPR of mice sensitized with 106 SRBC showed considerable perivascular cell infiltration predominantly consisting of mononuclear cells in dermis and hypodermis. However, contamination of polymorphonuclear leucocytes was inevitable in this lesion. There were polymorphs which infiltrated rather diffusely and macrophages with debris of SRBC in their cytoplasm around the depot of SRBC in the skin lesion besides perivascular infiltration. The footpad at 12 h showed only a small number of polymorphs and mononuclear cells scattered around the depot of SRBC in the skin.
Transferred cells
Thymus -
106
107 108
Marrow No. of mice 24 h FPR 2x 107 2x 107 2x 107 2x 107
4 5 5 5
1-0+0-4 3-2+0-8 4 4+0 5 5 8+0 5
Male C57BL mice were thymectomized at the age of 2 months, given total body irradiation (800 rad) and transferred with normal syngeneic lymphoid cells 12 days later. The mice were then sensitized intravenously with 106 SRBC 3 days thereafter. FPR at 24 h was measured in the fourth day
challenged mice.
Table 3. Suppressive effect of ferritin DH to SRBC
on
Treatment
No. of mice
24 h FPR
Ferritin
5 5
14 0+0-8 0-2+0-2
Male BDF1 mice aged 6 weeks were sensitized intravenously with 106 SRBC, and injected intraperitoneally with ferritin (0-2 mg) immediately before footpad challenge of SRBC on the fourth day of sensitization.
368
A. Mitsuoka et al. C57BL
BALjB/c
AKR
E
C3H
-R
0
-2 IT
2
-b 3
en
c) CL 10
a)
lCo
10"
Sensitizinq dose
Figure 4. Strain and age related differences in DH (histogram) and serum HA titre (hatched line) at 24 h after challenge. Male C57BL, AKR and C3H mice and female BALB/c mice were sensitized with varying doses of SRBC given intravenously 4 days previously. Numbers on the top of the columns indicate number of mice per group.
Pretreatment of mice with 0-2 mg of ferritin almost completely suppressed the infiltration of macrophages and polymorphs in the challenged site. Only a small number of lymphocytes was observed diffusely in the lesion. When toluidine blue staining was used for the detection of basophils and/or tissue mast cells which contain metachromatic cytoplasmic granules, these cells proved to be less than a few per cent among all nucleated cells infiltrating the lesion at 24 h. This finding was quite similar to that in the tuberculin type DH to methylated human serum albumin (MHSA) (unpublished observations). These cells were considered to be mast cells as concentration in this lesion was comparable to those in the nonchallenged footpads. Alkaline Giemsa staining after the method described by Dvorak et al. (1970) confirmed the absence of basophil infiltration in the lesion.
Effect of strain difference and ageing of mice on DH to SRBC Strain difference and ageing are well-known factors affecting immune responses. These factors were investigated in the DH to SRBC in comparison with humoral antibody production. Adult mice of relative young age (2-4 months) or old age (7-8 months) of several strains were sensitized with varying doses of SRBC, and challenged 4 days later (Fig. 4). Intensity of DH in several strains of mice was as follows; C57BL > AKR > BALB/c > C3H, that is to say, C57BL is a high responder, AKR and BALB/c are moderate responders and C3H is a low responder in DH. In general, optimal sensitizing dose of SRBC for DH was 106 in young mice, whereas it was 107 in old mice. In addition, optimal dose was also 106 and not 105 in mice as young as 4 weeks (data not in
Delayed hypersensitivity to sheep erythrocytes
figures). The only exception was BALB/c mice showing no age differences in DH, and such corresponds to observations in the DH to MHSA (Morikawa et al., in preparation). There was no definite tendency observed in the serum HA titre within different strains of mice tested.
DISCUSSION The time course of the DH reactivity after intravenous sensitization with 106 SRBC revealed the optimal sensitization-challenge interval to be 4 days, this result being somewhat different from the observation by Lagrange, Mackaness & Miller (1974) who found increasing DH reactivity from 4 days to beyond 6 days. We found that in the fourth day challenged mice, the DH reaction became apparent at about 12 h, reached a peak at 24 h and subsequently diminished, although the intensity was still considerable even at 48 h. The DH could be distinguished separately from the Arthus reaction, and appeared to be tuberculin type DH. The intensity of DH to SRBC was shown to be quantitatively dependent on the number of thymus cells transferred in the thymectomized and irradiated recipients. Kettman & Mathews (1975) reported that DH to SRBC induced by intravenous sensitization could be transferred into normal mice through anti-theta sensitive cells. An established DH state to SRBC was completely suppressed by a single injection of ferritin which was reported to be cytotoxic to peritoneal macrophages and non-cytotoxic to thymus or spleen cells in vitro (Baba et al., 1977). Hence, it is assumed that the DH to SRBC is a T-cell dependent phenomenon in both sensitization and effector stages and that it requires macrophages for manifestation. Histological findings also suggested that the DH to SRBC was substantially in the category of tuberculin type DH, in which perivascular cell infiltration was a constant finding in the lesion. Infiltrating cells dominantly consisted of mononuclear cells with polymorphs, and the population of basophils was negligible. Even though the DH to SRBC fits the concept of tuberculin-type DH, the only evident untoward feature was its rapid and transient reactivity after sensitization. This could be explained by prompt contact of SRBC antigen at a time to the immune device of the spleen due to direct injection of the antigen into the blood stream. Such might induce
369
immediate DH reactivity followed by rapid regulatory feedback of antibody production system or of certain suppressor cells. It has been discussed that sensitization without using CFA preferentially induces Jones-Mote DH but not tuberculin-type DH. The Jones-Mote DH, which is known to be also a thymus-dependent reaction (Richerson, Dvorak & Leskowitz, 1970), is characterized by its rapid development after challenge, not persisting after 48 h, and by its rapid and transient reactivity following sensitization in guineapigs (Nelson & Boyden, 1964). However, this reaction had been taken as a weak expression of DH as these characters themselves appeared not to have valid reason to discriminate this reaction from tuberculin type DH (see Turk, 1967). As reviewed by Turk (1975), the distinct entity of Jones-Mote DH in guinea-pigs appears to have been reinforced with ahistological feature of basophil dominant infiltration to the lesion, hence cutaneous basophil hypersensitivity (CBH) by Dvorak et al. (1970) and with evidences for a B-cell modulation of T-cell function in this reaction (Turk & Parker, 1973). However, the role of basophils on Jones-Mote DH in guinea-pigs is still obscure even at present (Askenase, 1976). In any case, the basophilic leucocytes appear to be normally absent from the blood in mice (Bloom & Fawcett, 1968). If it is assumed that infiltration of basophils in the skin lesion is an essential feature of the Jones-Mote type DH, the possibility of the presence of this reaction in mice is obscure in a descriptive histological sense. Turk and his co-workers detect skin reactions, in guinea-pigs, preferentially by measuring induration. However according to a typical description of CBH in guinea-pigs, a constant detectable change is erythema without increased vascular permiability accompanied by little or no induration and without requirement for macrophages for manifestation (Richerson et al., 1970). On the other hand, as in our case, footpad assay in mice permits detection of only factors relevant to induration, and in fact we demonstrated the requirement for macrophages in the DH to SRBC. These considerations lead to the conclusion that the Jones-Mote reaction was not demonstrated in mice. As for a B-cell modulation of DH reaction, it is obviously important in the DH in mice induced by intravenous sensitization with SRBC. However, it is true only when the sensitizing dose is much higher than 106 (Lagrange, Mackaness & Miller, 1974).
A. Mitsuoka et a1.
370
Finally, the DH to SRBC showed a distinct tendency to differ with the strain and age of the mice. Such a finding is significant as this tendency is similar to that observed in the tuberculin-type DH to MHSA (Morikawa et al., in preparation). On the other land, the serum HA titre at the time of DH measurement showed no significant strain and age difference. These results collectively support the concept that the DH induced by intravenous sensitization with low dose of SRBC is a phenomenon belonging to the category of tuberculin-type DH. The intrinsic adjuvanticity of the cell wall components of SRBC may play a role in inducing strong DH reactivity without the help of FCA. This supposed adjuvanticity is under investigation.
ACKNOWLEDGMENTS
Gratitude is due to Dr M. Itoh for pertinent advice and Dr T. Takeda and Dr K. Ohkawa for help with histological preparation. Thanks are also due to M. Matsushita, K. Kogishi and Y. Okumura for excellent technical assistance, and to M. Ohara for help with manuscript. This work was supported in part by grants from the Ministry of Health and Welfare and the Ministry of Education, Science and Culture, Japan. REFERENCES ASKENASE P.W. (1976) Cutaneous basophil hypersensitivity uncovered in the cell transfer of classical tuberculin hypersensitivity. J. Immunol. 117, 741. BABA M., HARADA T. & MORIKAWA S. (1977) Studies on delayed hypersensitivity in mice. I. Physicochemical and biological properties of preferential antigens for developing delayed hypersensitivity in mice. Acta pathol. jap. 27, 165. BLOOM W. & FAWCETr D.W. (1968) A Textbook of Histology.
Saunders, Philadelphia. CROWLE A.J. (1975) Delayed hypersensitivity in the mouse. Ad6anc. Immunol. 20, 197. CUNNINGHAM A.J. & SZENBERG A. (1968) Further improvement in the plaque technique detecting single antibody forming cells. Immunology, 14, 599.
DVORAK H.F., DVORAK A.M., SIMPSON B.A., RICHERSON H.B., LESKOWrrZ S. & KARNOVSKY M.J. (1970) Cutaneous basophil hypersensitivity. II. A light and electron microscopic description. J. exp. Med. 132, 558. KERCKHAERT J.A.M. (1974) Influence of cyclophosphamide on the delayed hypersensitivity in the mouse after intraperitoneal immunization. Ann. Immunol. 125, 559. KETrMAN J. (1972) Delayed hypersensitivity: is the same population of thymus derived cells responsible for cellular immunity reactions and the carrier effect? Immunol. Commun. 1, 289. KETTMAN J. & MATHEWS M.C. (1975) Radioresistance of cells responsible for delayed hypersensitivity reactions in the mouse. J. Immunol. 115, 606. LAGRANGE P.H., MACKANESS G.B. & MILLER T.E. (1974) Influence of dose and route of antigen injection on the immunological induction of T cells. J. exp. Med. 139, 528. LAGRANGE P.H., MACKANESS G.B. & MILLER T.E. (1974) Potentiation of T-cell-mediated immunity by selective suppression of antibody formation with cyclophosphamide. J. exp. Med. 139, 1529. MILLER T.E., MACKANESS G.B. & LAGRANGE P.H. (1973) Immunopotentiation with BCG. II. Modulation of the response to sheep red blood cells. J. nat. Cancer Inst. 51, 1669. NELSON D.S. & BOYDEN S.V. (1964) The cutaneous reactivity of guinea pigs to pure protein antigens. I. A critical evaluation of methods for the induction of delayed-type hypersensitivity to pure protein. Int. Arch. Allergy, 25, 279. NELSON D.S. & MILDENHALL P. (1967) Studies on cytophilic antibodies. I. The production by mice of macrophage cytophilic antibodies to sheep erythrocytes: relationship to the production of other antibodies and the development of delayed-type hypersensitivity. Aust. J. exp. Biol. med. Sci. 45, 113. RAFFEL S. & NEWEL J.M. (1958) The 'delayed hypersensitivity' induced by antigen-antibody complexes. J. exp. Med. 132, 823. RICHERSON H.B., DVORAK H.F. & LESKOWITZ S. (1970) Cutaneous basophil hypersensitivity. I. A new look at the Jones-Mote reaction, general characteristics. J. exp. Med. 132, 546. TURK J.L. (1967) Delayed Hypersensitivity. Frontiers of biology, Volume 4. North-Holland, Amsterdam. TURK J.L. (1975) Delayed Hypersensitivity, 2nd. edn. Frontiers of biology, volume 4. North-Holland, Amsterdam. TURK J.L. & PARKER D. (1973) Further studies on B-lymphocytes suppression in delayed hypersensitivity, indicating a possible mechanism for Jones-Mote hypersensitivity. Immunology, 24, 751.
Delayed hypersensitivity in mice induced by intravenous sensitization with sheep erythrocytes: evidence for tuberculin type delayed hypersensitivity of the reaction A. MITSUOKA, T. TERAMATSU, M. BABA,*t S. MORIKAWA*t & K. YASUHIRA* Department of Thoracic Surgery and *Department of Pathology, Chest Disease Research Institute, Kyoto University, Kyoto 606, Japan
Received 10 January 1977; acceptedfor publication 8 August 1977
Summary. Delayed hypersensitivity (DH) reaction
cutaneous sensitization with methylated human serum albumin (MHSA) in FCA. Taken collectively, it was concluded that this DH reaction can be categorized as the tuberculin type.
can be induced in mice by intravenous sensitization with sheep erythrocytes (SRBC). However, as the
sensitizing procedure is quite different from a usual mode of sensitization for DH using complete Freund's adjuvant (FCA), the nature of this reaction
INTRODUCTION
has been a matter of controversy. In an attempt to characterize this reaction, we placed special interest on two possibilities regarding the nature of this reaction; Jones-Mote reaction or tuberculin type DH. From the kinetics study on the DH after challenge, the DH reaction to SRBC in mice by intravenous sensitization was clearly distinguished from the Arthus reaction. The dose-response pattern of this reaction also suggested that the contribution of Arthus reactivity to delayed reactivity was negligible. Cell reconstitution experiments revealed this DH to be quantitatively thymus cell dependent. Furthermore, this DH required macrophages at its manifestation stage, and appearance of basophil infiltration at the lesion was absent. In addition, strain difference and ageing of host mice influenced the DH reaction in exactly the same fashion in which these factors influence the tuberculin type-DH induced by sub-
Delayed hypersensitivity (DH) to sheep erythrocytes (SRBC) in mice was reported for the first time by Nelson & Mildenhall (1967), who used a subcutaneous or intraperitoneal sensitization and the footpad test. According to their observations, sensitization of mice with SRBC could induce a strong DH reaction when Freund's complete adjuvant (FCA) was the sensitizing carrier, and slight DH reaction when Freund's incomplete adjuvant (IFA) or saline was the carrier. The DH to SRBC induced without FCA was considered to be a Jones-Mote reaction as described by Raffel & Newel (1958), and tuberculin-type DH was considered to develop only by sensitization with FCA when the antigens were soluble proteins or heterologous erythrocytes as reviewed by Crowle (1975). Recently, the development of DH in mice to SRBC was again observed by Miller, Mackaness & Lagrange (1973), who used a subcutaneous sensitization with SRBC in saline. Intraperitoneal sensitization with SRBC in saline also appeared to produce a considerable skin DH in mice (Kerckhaert, 1974). One of the most interesting findings was
t Present address: Department of Pathology, Shimane Medical School, Izumo 693, Japan. Correspondence: Dr Akio Mitsuoka, Department of Thoracic Surgery, Chest Disease Research Institute, Kyoto University, Kyoto 606, Japan.
363
364
A. Mitsuoka et al.
the demonstration of the ability of mice to develop DH after intravenous sensitization with SRBC in saline (Kettman, 1972). These reactions have been currently used in studies of the immune regulation involved in DH. However, based on the rapid kinetics of the reactions, these phenomena may also be considered a Jones-Mote reaction different from the tuberculin-type DH. In this report, the nature of the DH in mice, as induced by intravenous sensitization with SRBC, was investigated in an attempt to clarify whether such belongs to the tuberculin-type of Jones-Mote type DH.
MATERIALS AND METHODS Mice AKR/J BALB/c Crgl, and C3H/HeMs mice bred and fed in the animal house of CDRI, Kyoto University, and C57BL/6 and BDF1 mice from the Experimental Animal Cooperative Association of Shizuoka (Hamamatsu) were used. Detailed records included age and sex of mice are described in the text or the footnotes.
Antigen SRBC were obtained in Alsever's solution from the Nikken Animal Blood Supply Centre (Kyoto). These cells were stored at 0-4' for less than 30 days and were washed three times and suspended in phosphate buffered saline (PBS, pH 7-1) before use. Sensitization
Mice were sensitized by an intravenous administration into the retro-ocular plexus of a solution of 0-2 ml consisting of varying doses of SRBC. Assay for skin reaction A challenge injection of about 108 SRBC in 0-02 ml of PBS was given into the right footpad at various intervals after sensitization. As a standard schedule for DH, the interval of 4 days was adopted for reasons described later. The method for measurement of the footpad reaction (FPR) was fully described in our previous paper (Baba, Harada & Morikawa, 1977). In most experiments, thickness of both the right and left foot was measured at 24 h after challenge just following exanguination, and the difference between these two measurements was described in 1/10 mm (FPR) as the intensity of
DH. In some experiments, FPR was measured at different intervals after challenge other than 24 h.
Assay for antibody producing cells The number of IgM-plaque forming cells (PFC) against SRBC of the spleen was assayed by the method of Cunningham & Szenberg (1968). Titration of serum antibody Peripheral blood was obtained just before measurement of FPR. Serum was separated from the blood, pooled from each group of mice and stored at - 1520° until assay. Titration of haemagglutinin (HA) and haemolysin (HL) of the serum was performed using microtitration trays.
Histological methods After the intensity of FPR was measured, the footpad was excised and fixed in 10% formalin solution. The paraffin section was stained with haematoxylin and eosin. For the detection of possible infiltration of basophils, tissues were fixed in 5 % phosphate buffered glutaraldehyde (pH 7 3), postfixed in osmium tetroxide, and 1 pm epon embedded section were prepared. These sections were stained for 1 h in Giemsa solution diluted 1:10 in 2% sodium borate solution after the method by Dvorak, Dvorak, Simpson, Richerson, Leskowitz & Karnovski (1970). Alternatively, tissues were fixed in 10% formalin solution containing 1 % cetylpyridium chloride, the procedure of which can insolubilize heparin-containing metachromatic granules of basophils or mast cells. The fixed tissues were embedded in paraffin and the sections were stained with toluidine blue. Pretreatments of mice In one experiment, mice were thymectomized at the age of 2 months, and given a total body irradiation of 800 rad 12 days later. These mice were then reconstituted with the proper number of lymphoid cells on the same day of irradiation. The syngeneic donor mice were 4 weeks of age for thymus cell transfer, or 8 months for bone marrow cell transfer. The recipient mice were all checked after FPR measurement to confirm that the thymus had been totally excised. In another experiment, mice were injected intraperitoneally with 0-2 ml of 0-1 % ferritin solution. The ferritin solution was made by resolving
six times-crystallized ferritin from horse spleen (Pentex Biochemicals, Kankakee, U.S.A.) in PBS.
365
Delayed hypersensitivity to sheep erythrocytes RESULTS
Time effect on the development of DH to SRBC after sensitization Male C57BL mice aged 2 or 3 months were sensitized intravenously with 106 SRBC and given a challenge injection at varying intervals thereafter. FPR at 24 h after challenge was measured (Fig. 1). The optimal interval between sensitization and challenge was 4 days in group of either age. In the case of higher dose sensitization, the peak response was seen also on the fourth day, but the response was less intense (data not in figures).
14
12 I I
I I
10
I
I I
F
I
I
6
-
k
I
I
c-JIT
I
8
I1
I
I
I I I
Kinetics of FPR to SRBC after challenge Kinetics of FPR after challenge were investigated in mice sensitized with 106 SRBC intravenously at 2 months old (Fig. 2) or 3 months old (Fig. 3). In Fig. 2, a slight response was observed at 3 h and a prominent peak was seen at 24 h on the fourth day of challenge. When the mice were challenged 6 or 8 days after sensitization, a higher peak response at 3 h and a lower response at 24 h were seen. The peak response was seen to be delayed more than 24 h on the third dayofchallenge. TheFPRat 58h differed among each group, despite a similar serum HA titre. Similarly in Fig. 3, a marked peak response was observed at 24 h in the fourth day group. It is noted that the DH reaction in this group became apparent after 12 h and was still considerable at 48 h. A plateau reaction which increased from 24 to 48 h and subsequently diminished was observed in the third day group. Of interest is the fact that the Arthus reaction was different in intensity as seen in Figs 2 and 3, whereas the DH intensity showed a quite reproducible pattern in both cases (see Fig. 1). From these results, DH is apparently not influenced by the Arthus reaction, particularly in mice challenged on the fourth day. Thus, in further studies, we adopted the interval of 4 days for investigation of the DH to SRBC. Influence of sensitizing dose on DH and antibody production Male C57BL mice aged 3 5 months were sensitized with varying doses of SRBC and challenged 4 days later. FPR of each mouse was measured at 3 and 24 h after challenge. Serum HA and HL titres and splenic PFC were also assayed (Table 1).
I I -q Ln
6
I i
I
I
I \
4
T~~~~~~~~~~~~~4
-.I-
2
3
4
5 6 after sensitization Days
7
8
Figure 1. DH to SRBC in mice at various intervals after sensitization. Male C57BL mice aged 2 (0) or 3 (0) months were sensitized intravenously with 106 SRBC. FPR was measured at 24 h after challenge. Each group included five or more mice. Vertical bars are standard errors of the means.
A direct correlation was found between the sensitizing dose and Arthus reaction as well as PFC, HA and HL titres, particularly when the dose was over 106. On the other hand, as DH was the highest at the dose of 106, the presence of an optimal sensitizing dose in DH was confirmed.
Thymus dependency of DH to SRBC Male C57BL mice were thymectomized at the age of 2 months, 800 rad irradiated and reconstituted with lymphoid cells from normal syngeneic male mice. The recipient mice were then sensitized with 106 SRBC given intravenously followed by challenge 4
days thereafter.
366
A. Mitsuoka et al. IC'
E
6.0
5-5 63 U-~
~
~
17 ~
~
~
~
245 ~~~~~~~~~~~~-
2
-~~~~~~~~~~~~~~~~-
17
3 6
24
58
Hours after challenge
Figure 2. Kinetics of FPR to SRBC after challenge. Male C57BL mice aged 2 months were sensitized intravenously with 106 SRBC 3 (0-0), 4 (0-0), 6 (O - - 0) or 8 ( -- 0) days previously. Serum HA titre (log 2) at 58 h after challenge is also shown. Each group included five or more mice.
E8 6
4 0
;
3
/
12
24
48
72
Hours after challenge
Figure 3. Kinetics of FPR to SRBC after challenge. Male C57BL mice aged 3 months were sensitized intravenously with 106 SRBC 3 (0-0), 4 (0-0), 5 (O - - 0), or 6 ( -- 0) days previously. Each group included five or more mice.
Delayed hypersensitivity
to
sheep erythrocytes
367
Table 1. Delayed hypersensitivity and antibody production to varying doses of SRBC in mice
Antibody titret 3 h FPR*
Dose -
3-5+ 1-5 2-8+P19
05 106 107
3-8+2-7 4-2+ 1-7 5 0+ 2-4 6-2+ 13
lo8
109
24 h FPR* 3 0+2-0 7-6+ 1 7 15-4+3-2 10-7+ 1 9 3-4+ 1-6 1-2+ 10
PFC/spleent (x 100) 3-7
3-2 99 250 368 581
HA
HL
1.0 1.0 2-0 50 70 7-0
0 0 45 6-0 8-0 9-0
Male C57BL mice aged 3-5 months were sensitized intravenously with varying doses of SRBC, and challenged 4 days later. * Mean + standard error (1/10 mm). t Spleen cells and serum were obtained from mice at 24 h after challenge. Each group included five mice.
Table 2 shows that the intensity of DH increased in proportion to the number of thymus cells transferred together with a given number of bone marrow cells. Mice not given thymocytes did not develop detectable DH. Effect of fefritin on DH to SRBC In our previous work (Baba et al., 1977), ferritin was revealed to be as selectively toxic to macrophages as was carragenan. To determine the participation of macrophages in eliciting delayed footpad swelling, male BDF1 mice aged 6 weeks sensitized with 106 SRBC were given 0-2 mg of ferritin intraperitoneally just before footpad challenge on the
Table 2. T cell dependence of DH to SRBC in mice
fourth day of sensitization. As shown in Table 3, DH reaction was completely inhibited, while this treatment appeared to have no effect on the general condition of mice.
Histological studies on FPR to SRBC Histology of 24 h FPR of mice sensitized with 106 SRBC showed considerable perivascular cell infiltration predominantly consisting of mononuclear cells in dermis and hypodermis. However, contamination of polymorphonuclear leucocytes was inevitable in this lesion. There were polymorphs which infiltrated rather diffusely and macrophages with debris of SRBC in their cytoplasm around the depot of SRBC in the skin lesion besides perivascular infiltration. The footpad at 12 h showed only a small number of polymorphs and mononuclear cells scattered around the depot of SRBC in the skin.
Transferred cells
Thymus -
106
107 108
Marrow No. of mice 24 h FPR 2x 107 2x 107 2x 107 2x 107
4 5 5 5
1-0+0-4 3-2+0-8 4 4+0 5 5 8+0 5
Male C57BL mice were thymectomized at the age of 2 months, given total body irradiation (800 rad) and transferred with normal syngeneic lymphoid cells 12 days later. The mice were then sensitized intravenously with 106 SRBC 3 days thereafter. FPR at 24 h was measured in the fourth day
challenged mice.
Table 3. Suppressive effect of ferritin DH to SRBC
on
Treatment
No. of mice
24 h FPR
Ferritin
5 5
14 0+0-8 0-2+0-2
Male BDF1 mice aged 6 weeks were sensitized intravenously with 106 SRBC, and injected intraperitoneally with ferritin (0-2 mg) immediately before footpad challenge of SRBC on the fourth day of sensitization.
368
A. Mitsuoka et al. C57BL
BALjB/c
AKR
E
C3H
-R
0
-2 IT
2
-b 3
en
c) CL 10
a)
lCo
10"
Sensitizinq dose
Figure 4. Strain and age related differences in DH (histogram) and serum HA titre (hatched line) at 24 h after challenge. Male C57BL, AKR and C3H mice and female BALB/c mice were sensitized with varying doses of SRBC given intravenously 4 days previously. Numbers on the top of the columns indicate number of mice per group.
Pretreatment of mice with 0-2 mg of ferritin almost completely suppressed the infiltration of macrophages and polymorphs in the challenged site. Only a small number of lymphocytes was observed diffusely in the lesion. When toluidine blue staining was used for the detection of basophils and/or tissue mast cells which contain metachromatic cytoplasmic granules, these cells proved to be less than a few per cent among all nucleated cells infiltrating the lesion at 24 h. This finding was quite similar to that in the tuberculin type DH to methylated human serum albumin (MHSA) (unpublished observations). These cells were considered to be mast cells as concentration in this lesion was comparable to those in the nonchallenged footpads. Alkaline Giemsa staining after the method described by Dvorak et al. (1970) confirmed the absence of basophil infiltration in the lesion.
Effect of strain difference and ageing of mice on DH to SRBC Strain difference and ageing are well-known factors affecting immune responses. These factors were investigated in the DH to SRBC in comparison with humoral antibody production. Adult mice of relative young age (2-4 months) or old age (7-8 months) of several strains were sensitized with varying doses of SRBC, and challenged 4 days later (Fig. 4). Intensity of DH in several strains of mice was as follows; C57BL > AKR > BALB/c > C3H, that is to say, C57BL is a high responder, AKR and BALB/c are moderate responders and C3H is a low responder in DH. In general, optimal sensitizing dose of SRBC for DH was 106 in young mice, whereas it was 107 in old mice. In addition, optimal dose was also 106 and not 105 in mice as young as 4 weeks (data not in
Delayed hypersensitivity to sheep erythrocytes
figures). The only exception was BALB/c mice showing no age differences in DH, and such corresponds to observations in the DH to MHSA (Morikawa et al., in preparation). There was no definite tendency observed in the serum HA titre within different strains of mice tested.
DISCUSSION The time course of the DH reactivity after intravenous sensitization with 106 SRBC revealed the optimal sensitization-challenge interval to be 4 days, this result being somewhat different from the observation by Lagrange, Mackaness & Miller (1974) who found increasing DH reactivity from 4 days to beyond 6 days. We found that in the fourth day challenged mice, the DH reaction became apparent at about 12 h, reached a peak at 24 h and subsequently diminished, although the intensity was still considerable even at 48 h. The DH could be distinguished separately from the Arthus reaction, and appeared to be tuberculin type DH. The intensity of DH to SRBC was shown to be quantitatively dependent on the number of thymus cells transferred in the thymectomized and irradiated recipients. Kettman & Mathews (1975) reported that DH to SRBC induced by intravenous sensitization could be transferred into normal mice through anti-theta sensitive cells. An established DH state to SRBC was completely suppressed by a single injection of ferritin which was reported to be cytotoxic to peritoneal macrophages and non-cytotoxic to thymus or spleen cells in vitro (Baba et al., 1977). Hence, it is assumed that the DH to SRBC is a T-cell dependent phenomenon in both sensitization and effector stages and that it requires macrophages for manifestation. Histological findings also suggested that the DH to SRBC was substantially in the category of tuberculin type DH, in which perivascular cell infiltration was a constant finding in the lesion. Infiltrating cells dominantly consisted of mononuclear cells with polymorphs, and the population of basophils was negligible. Even though the DH to SRBC fits the concept of tuberculin-type DH, the only evident untoward feature was its rapid and transient reactivity after sensitization. This could be explained by prompt contact of SRBC antigen at a time to the immune device of the spleen due to direct injection of the antigen into the blood stream. Such might induce
369
immediate DH reactivity followed by rapid regulatory feedback of antibody production system or of certain suppressor cells. It has been discussed that sensitization without using CFA preferentially induces Jones-Mote DH but not tuberculin-type DH. The Jones-Mote DH, which is known to be also a thymus-dependent reaction (Richerson, Dvorak & Leskowitz, 1970), is characterized by its rapid development after challenge, not persisting after 48 h, and by its rapid and transient reactivity following sensitization in guineapigs (Nelson & Boyden, 1964). However, this reaction had been taken as a weak expression of DH as these characters themselves appeared not to have valid reason to discriminate this reaction from tuberculin type DH (see Turk, 1967). As reviewed by Turk (1975), the distinct entity of Jones-Mote DH in guinea-pigs appears to have been reinforced with ahistological feature of basophil dominant infiltration to the lesion, hence cutaneous basophil hypersensitivity (CBH) by Dvorak et al. (1970) and with evidences for a B-cell modulation of T-cell function in this reaction (Turk & Parker, 1973). However, the role of basophils on Jones-Mote DH in guinea-pigs is still obscure even at present (Askenase, 1976). In any case, the basophilic leucocytes appear to be normally absent from the blood in mice (Bloom & Fawcett, 1968). If it is assumed that infiltration of basophils in the skin lesion is an essential feature of the Jones-Mote type DH, the possibility of the presence of this reaction in mice is obscure in a descriptive histological sense. Turk and his co-workers detect skin reactions, in guinea-pigs, preferentially by measuring induration. However according to a typical description of CBH in guinea-pigs, a constant detectable change is erythema without increased vascular permiability accompanied by little or no induration and without requirement for macrophages for manifestation (Richerson et al., 1970). On the other hand, as in our case, footpad assay in mice permits detection of only factors relevant to induration, and in fact we demonstrated the requirement for macrophages in the DH to SRBC. These considerations lead to the conclusion that the Jones-Mote reaction was not demonstrated in mice. As for a B-cell modulation of DH reaction, it is obviously important in the DH in mice induced by intravenous sensitization with SRBC. However, it is true only when the sensitizing dose is much higher than 106 (Lagrange, Mackaness & Miller, 1974).
A. Mitsuoka et a1.
370
Finally, the DH to SRBC showed a distinct tendency to differ with the strain and age of the mice. Such a finding is significant as this tendency is similar to that observed in the tuberculin-type DH to MHSA (Morikawa et al., in preparation). On the other land, the serum HA titre at the time of DH measurement showed no significant strain and age difference. These results collectively support the concept that the DH induced by intravenous sensitization with low dose of SRBC is a phenomenon belonging to the category of tuberculin-type DH. The intrinsic adjuvanticity of the cell wall components of SRBC may play a role in inducing strong DH reactivity without the help of FCA. This supposed adjuvanticity is under investigation.
ACKNOWLEDGMENTS
Gratitude is due to Dr M. Itoh for pertinent advice and Dr T. Takeda and Dr K. Ohkawa for help with histological preparation. Thanks are also due to M. Matsushita, K. Kogishi and Y. Okumura for excellent technical assistance, and to M. Ohara for help with manuscript. This work was supported in part by grants from the Ministry of Health and Welfare and the Ministry of Education, Science and Culture, Japan. REFERENCES ASKENASE P.W. (1976) Cutaneous basophil hypersensitivity uncovered in the cell transfer of classical tuberculin hypersensitivity. J. Immunol. 117, 741. BABA M., HARADA T. & MORIKAWA S. (1977) Studies on delayed hypersensitivity in mice. I. Physicochemical and biological properties of preferential antigens for developing delayed hypersensitivity in mice. Acta pathol. jap. 27, 165. BLOOM W. & FAWCETr D.W. (1968) A Textbook of Histology.
Saunders, Philadelphia. CROWLE A.J. (1975) Delayed hypersensitivity in the mouse. Ad6anc. Immunol. 20, 197. CUNNINGHAM A.J. & SZENBERG A. (1968) Further improvement in the plaque technique detecting single antibody forming cells. Immunology, 14, 599.
DVORAK H.F., DVORAK A.M., SIMPSON B.A., RICHERSON H.B., LESKOWrrZ S. & KARNOVSKY M.J. (1970) Cutaneous basophil hypersensitivity. II. A light and electron microscopic description. J. exp. Med. 132, 558. KERCKHAERT J.A.M. (1974) Influence of cyclophosphamide on the delayed hypersensitivity in the mouse after intraperitoneal immunization. Ann. Immunol. 125, 559. KETrMAN J. (1972) Delayed hypersensitivity: is the same population of thymus derived cells responsible for cellular immunity reactions and the carrier effect? Immunol. Commun. 1, 289. KETTMAN J. & MATHEWS M.C. (1975) Radioresistance of cells responsible for delayed hypersensitivity reactions in the mouse. J. Immunol. 115, 606. LAGRANGE P.H., MACKANESS G.B. & MILLER T.E. (1974) Influence of dose and route of antigen injection on the immunological induction of T cells. J. exp. Med. 139, 528. LAGRANGE P.H., MACKANESS G.B. & MILLER T.E. (1974) Potentiation of T-cell-mediated immunity by selective suppression of antibody formation with cyclophosphamide. J. exp. Med. 139, 1529. MILLER T.E., MACKANESS G.B. & LAGRANGE P.H. (1973) Immunopotentiation with BCG. II. Modulation of the response to sheep red blood cells. J. nat. Cancer Inst. 51, 1669. NELSON D.S. & BOYDEN S.V. (1964) The cutaneous reactivity of guinea pigs to pure protein antigens. I. A critical evaluation of methods for the induction of delayed-type hypersensitivity to pure protein. Int. Arch. Allergy, 25, 279. NELSON D.S. & MILDENHALL P. (1967) Studies on cytophilic antibodies. I. The production by mice of macrophage cytophilic antibodies to sheep erythrocytes: relationship to the production of other antibodies and the development of delayed-type hypersensitivity. Aust. J. exp. Biol. med. Sci. 45, 113. RAFFEL S. & NEWEL J.M. (1958) The 'delayed hypersensitivity' induced by antigen-antibody complexes. J. exp. Med. 132, 823. RICHERSON H.B., DVORAK H.F. & LESKOWITZ S. (1970) Cutaneous basophil hypersensitivity. I. A new look at the Jones-Mote reaction, general characteristics. J. exp. Med. 132, 546. TURK J.L. (1967) Delayed Hypersensitivity. Frontiers of biology, Volume 4. North-Holland, Amsterdam. TURK J.L. (1975) Delayed Hypersensitivity, 2nd. edn. Frontiers of biology, volume 4. North-Holland, Amsterdam. TURK J.L. & PARKER D. (1973) Further studies on B-lymphocytes suppression in delayed hypersensitivity, indicating a possible mechanism for Jones-Mote hypersensitivity. Immunology, 24, 751.
