Immunology 1977 32 645
Tumour-associated inhibition of immediate hypersensitivity reactions in mice
N. R. LYNCH & J. C. S ALO MON Laboratoire d'Immunopathologie, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France
Received 28 June 1976; accepted for publication 24 September 1976
Summary. The intensity of anaphylactic shock was lower in C3H mice carrying a methylcholanthreneinduced tumour (McC3) than in their normal counterparts when immunized with ovalbumin and challenged i.v. after 14 days. This tumour-associated inhibitory effect on active systemic anaphylaxis was exerted mainly on events occurring after homocytotropic antibody synthesis because the serum titres of these antibodies were comparable in normal and tumour-bearing animals. In addition, passive systemic anaphylactic reactions were suppressed in animals carrying the tumour and the sensitivity of these animals to challenge with histamine and serotonin mixtures was also reduced. The presence of a growing McC3 tumour did not, however, diminish the amine-sensitizing effect of treatment with Bordetella pertussis vaccine. The McC3 tumour inhibited the generation of passive cutaneous anaphylactic reactions, an effect that was also exerted by a tumour extract, particularly when administered to the recipients shortly before antigen challenge. Thus immediate hypersensitivity reactions, like a variety of other immunological processes, can be inhibited by tumour products which by compromising the immune status of the host might permit tumour growth. The nature of the inhibiting factor is unknown, except that it is probably not the amine-degrading enzyme histaminase. In addition, while it is uncertain whether the Correspondence: Dr N. R. Lynch, Laboratoire d'Immunopathologie, Institut de Recherches Scientifiques sur le Cancer, 94800 Villejuif, France.
645
inhibitory effect is exerted directly or indirectly, the possible importance of prostaglandins in the phenomenon is discussed. INTRODUCTION The suppression of immunological responsiveness has been demonstrated in a variety of situations, including neoplasia (Weigle, 1973). Tumours have been found to inhibit antibody synthesis (Kamo, Patel, Kateley & Friedman, 1975; Plescia, Smith & Grinwich, 1975) macrophage accumulation or phagocytosis (Fauve, Hevin, Jacob, Gaillard & Jacob, 1974; Snyderman, Pike, Blaylock & Weinstein, 1976; North, Kirstein & Tuttle, 1976) and antigen or mitogen stimulation of lymphocytes (Levy, Smith, Whitney, McMaster & Kilburn, 1976). It is possible that the existence of such inhibitory activities is related to the ability of tumours to grow in an otherwise immunologically competent host (Plescia et al., 1975). Although the physiological role of the immediate hypersensitivity or anaphylactic reaction (IHR) is uncertain, there is evidence to suggest an involvement in the resistance to parasitic infection (Barth, Jarrett & Urquhart, 1966; Bloch, Cygan & Waltin, 1972; Dean, Murrell, Minard & Vannier, 1976). It has been proposed that IgE antibody acts as a 'gatekeeper', whereby the enhanced vascular permeability resulting from IHR in the vicinity of an invading organism allows greater access of effector immune cells or antibodies
646
N. R. Lynch & J.-C. Salomon
to the site of infection (Steinberg, Ishizaka & Norman, 1974). It has been found that i.v. injected dyes do not effectively penetrate into tumours (Goldacre & Sylven, 1959) and thus has been suggested that tumours are somewhat isolated from the circulating blood components of their hosts. It is possible, therefore, that IHR assist in anti-tumour reactions by enhancing the access of the immune system to tumour foci in the process of establishment. An extension of this reasoning provides the basis for the work described here, where the possibility that an actively growing tumour might inhibit IHR was investigated. IHR result from the interaction of antigen with specific homocytotropic antibodies at the surface of mast cells and the subsequent release of autopharmacologic mediators such as histamine or serotonin (Vaz & Prouvost-Danon, 1969). The possibility was therefore investigated that tumour growth might modulate the intensity of IHR, using active and passive systemic anaphylaxis and passive cutaneous anaphylaxis as indicators in tumourbearing animals. The possible involvement of IHR in tumour rejection processes will form the basis of a later communication.
MATERIALS AND METHODS Mice The C3H and Swiss mice used in these experiments were of both sexes and between 4 and 12 weeks of age. Experimental groups were matched for age and sex. They were bred in the barrier-maintained specific pathogen-free animal facilities of the IRSC, Villejuif, France. Tumour The McC3 tumour was originally induced in a C3H mouse by the i.m. injection of methylcholanthrene and was maintained by serial subcutaneous trocar transplantation and stored frozen. Tumour extracts were prepared by homogenizing 5g of non-necrotic tumour tissue in 10 ml of normal saline then ultracentrifugation at 75,000 g for 90 min. Mice bearing large McC3 tumours (> 20 mm mean diameter) were bled to provide tumour-modified serum. Anti-ovalbumin antisera (anti-OA) Female DBA/1 mice were immunized i.p. with
1
ug OA (Sigma) adsorbed to A1(OH)3 gel, three times at monthly intervals and bled 15 days after the last injection. C3H and Swiss mice were immunized i.p. with 50 ,ug OA adsorbed to 5 mg A1(OH)3 gel and bled after 14 days. The short-term immunization permitted the investigation of the effect of small tumours on active systemic anaphylaxis.
Passive cutaneous anaphylactic reactions (PCA) The dorsal skin of mice was shaved and 30 ,l of serial dilutions of antiserum injected i.d. After either 2 h or 48 h (i.e. the latent period, used to detect IgGl or IgE activity respectively) 100 ,ug of OA was injected i.v. in 0 2 ml of 0 5 per cent Evans blue dye. The intensities of the PCA reactions were scored on an arbitrary 0-4 scale and the PCA titre was defined as the reciprocal of the serum dilution giving threshold reactions in at least two of three recipients. PCA reactions in McC3-bearing C3H mice were performed in the dorsal skin, while the tumour was always grafted ventrally. Haemoconcentration The technique utilized was basically that of Iff & Vaz (1966). Small volumes of blood (5 ,ul) were taken from mice by bleeding from the orbital plexus before anaphylactic challenge, then 5 and 10 min after. The samples were diluted in 2 5 ml of 1 per cent Na2CO3 and the optical density at 541 nm read. The severity of hypovolaemic shock was assessed by the percentage increase in blood haemoglobulin concentration after challenge. Challenges with histamine and serotonin mixtures were also performed 4 days after the i.p. injection of 1010 merthiolate-killed B. pertussis organisms (J. Raynaud Institute Pasteur). This treatment greatly increases the sensitivity of normal animals to the action of vasoactive amines (Munoz & Bergman, 1968). Histamine assay Serum histaminase levels were assayed by the technique of Roscoe & Kupfer (1972) as modified by Lin, Orcutt & Stolbach (1975) where, after incubation of 14C-labelled histamine with the serum, the deaminated reaction products were separated from the unreacted parent molecule on phosphocellulose and then quantified by ,B counting. Also, to test the anti-tumour activity of histaminase inhibitors, C3H mice were treated with aminoguanidine hemi-sulphate (Sigma) in their drinking water at a level of 20 ug/ml for a week before
Immediate hypersensitivity reactions in mice grafting with McC3 and then for the following 6 weeks. Tumour diameters were measured twice weekly. RESULTS Anaphylactic challenge of C3H mice bearing McC3 tumours
Normal or McC3 tumour-bearing (10-20 mm diameter) C3H mice were anaphylactically challenged by the means described below, and the severity of hypovolaemic shock assessed by quantifying the resulting haemoconcentration. In general, animals with larger McC3 tumours (> 20 mm) could not be used because these mice were severely anaemic, with very low starting haemoglobin concentrations. Inconsistent results were obtained with such animals. Active systemic anaphylaxis (ASA) was induced by sensitization with OA adsorbed to A1(OH)3 gel and i.v. challenge with OA after 14 days. The animals with McC3 tumours had received grafts 2 days after sensitization and the anti-OA PCA titres in a pool of these day 14 sera were 320 for IgG1 and 80 for IgE, compared with 320 and 160 respectively in the control. The data presented in Table 1 demonstrate that the severity of ASA was significantly lower in McC3 tumour-bearing C3H mice than normal. The intensity of passive systemic anaphylaxis
647
(PSA) mediated by a DBA/1 anti-OA antiserum was also significantly lower in C3H mice carrying the McC3 tumour (Table 1). C3H mice with McC3 tumours were more resistant than normal to challenge with a mixture of the amines primarily responsible for anaphylactic shock, histamine and serotonin (Table 1). However, both normal and tumour-bearing C3H mice were sensitized to the vasoactivity of these amines by treatment with B. pertussis vaccine (Table 1). Histaminase levels were estimated in the serum of normal and tumour-bearing C3H mice but were bearly detectable, being < 0 05 ,g amine hydrolyzed/ ml/h, in both situations. Also, McC3 tumour grafts grew no less rapidly than usual in C3H mice receiving the histaminase inhibitor aminoguanidine in their drinking water. PCA titres in C3H mice bearing McC3 tumours The 2 h (IgG1) and 48 h (IgE) PCA titres of anti-OA antisera were determined in either normal C3H mice or C3H mice bearing McC3 tumours. Despite the dark pigmentation of the outer layer of C3H mice skin, the PCA reactions were well defined when the skin was reflected and the clear inner skin layer examined. The PCA titres were easily determined in normal C3H mice. In C3H mice carrying large (15-25 mm diameter) McC3 tumours the PCA reactions were diffusely ill-defined and the titres
Table 1. Anaphylactic challenge of C3H mice bearing McC3 tumours
Recipient*
Normal C3H C3H with McC3
Mean per cent haemoconcentration
ASAt
PSAt
H+ S§
B. pertussis; H+ S¶
26+4 P
Tumour-associated inhibition of immediate hypersensitivity reactions in mice
N. R. LYNCH & J. C. S ALO MON Laboratoire d'Immunopathologie, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France
Received 28 June 1976; accepted for publication 24 September 1976
Summary. The intensity of anaphylactic shock was lower in C3H mice carrying a methylcholanthreneinduced tumour (McC3) than in their normal counterparts when immunized with ovalbumin and challenged i.v. after 14 days. This tumour-associated inhibitory effect on active systemic anaphylaxis was exerted mainly on events occurring after homocytotropic antibody synthesis because the serum titres of these antibodies were comparable in normal and tumour-bearing animals. In addition, passive systemic anaphylactic reactions were suppressed in animals carrying the tumour and the sensitivity of these animals to challenge with histamine and serotonin mixtures was also reduced. The presence of a growing McC3 tumour did not, however, diminish the amine-sensitizing effect of treatment with Bordetella pertussis vaccine. The McC3 tumour inhibited the generation of passive cutaneous anaphylactic reactions, an effect that was also exerted by a tumour extract, particularly when administered to the recipients shortly before antigen challenge. Thus immediate hypersensitivity reactions, like a variety of other immunological processes, can be inhibited by tumour products which by compromising the immune status of the host might permit tumour growth. The nature of the inhibiting factor is unknown, except that it is probably not the amine-degrading enzyme histaminase. In addition, while it is uncertain whether the Correspondence: Dr N. R. Lynch, Laboratoire d'Immunopathologie, Institut de Recherches Scientifiques sur le Cancer, 94800 Villejuif, France.
645
inhibitory effect is exerted directly or indirectly, the possible importance of prostaglandins in the phenomenon is discussed. INTRODUCTION The suppression of immunological responsiveness has been demonstrated in a variety of situations, including neoplasia (Weigle, 1973). Tumours have been found to inhibit antibody synthesis (Kamo, Patel, Kateley & Friedman, 1975; Plescia, Smith & Grinwich, 1975) macrophage accumulation or phagocytosis (Fauve, Hevin, Jacob, Gaillard & Jacob, 1974; Snyderman, Pike, Blaylock & Weinstein, 1976; North, Kirstein & Tuttle, 1976) and antigen or mitogen stimulation of lymphocytes (Levy, Smith, Whitney, McMaster & Kilburn, 1976). It is possible that the existence of such inhibitory activities is related to the ability of tumours to grow in an otherwise immunologically competent host (Plescia et al., 1975). Although the physiological role of the immediate hypersensitivity or anaphylactic reaction (IHR) is uncertain, there is evidence to suggest an involvement in the resistance to parasitic infection (Barth, Jarrett & Urquhart, 1966; Bloch, Cygan & Waltin, 1972; Dean, Murrell, Minard & Vannier, 1976). It has been proposed that IgE antibody acts as a 'gatekeeper', whereby the enhanced vascular permeability resulting from IHR in the vicinity of an invading organism allows greater access of effector immune cells or antibodies
646
N. R. Lynch & J.-C. Salomon
to the site of infection (Steinberg, Ishizaka & Norman, 1974). It has been found that i.v. injected dyes do not effectively penetrate into tumours (Goldacre & Sylven, 1959) and thus has been suggested that tumours are somewhat isolated from the circulating blood components of their hosts. It is possible, therefore, that IHR assist in anti-tumour reactions by enhancing the access of the immune system to tumour foci in the process of establishment. An extension of this reasoning provides the basis for the work described here, where the possibility that an actively growing tumour might inhibit IHR was investigated. IHR result from the interaction of antigen with specific homocytotropic antibodies at the surface of mast cells and the subsequent release of autopharmacologic mediators such as histamine or serotonin (Vaz & Prouvost-Danon, 1969). The possibility was therefore investigated that tumour growth might modulate the intensity of IHR, using active and passive systemic anaphylaxis and passive cutaneous anaphylaxis as indicators in tumourbearing animals. The possible involvement of IHR in tumour rejection processes will form the basis of a later communication.
MATERIALS AND METHODS Mice The C3H and Swiss mice used in these experiments were of both sexes and between 4 and 12 weeks of age. Experimental groups were matched for age and sex. They were bred in the barrier-maintained specific pathogen-free animal facilities of the IRSC, Villejuif, France. Tumour The McC3 tumour was originally induced in a C3H mouse by the i.m. injection of methylcholanthrene and was maintained by serial subcutaneous trocar transplantation and stored frozen. Tumour extracts were prepared by homogenizing 5g of non-necrotic tumour tissue in 10 ml of normal saline then ultracentrifugation at 75,000 g for 90 min. Mice bearing large McC3 tumours (> 20 mm mean diameter) were bled to provide tumour-modified serum. Anti-ovalbumin antisera (anti-OA) Female DBA/1 mice were immunized i.p. with
1
ug OA (Sigma) adsorbed to A1(OH)3 gel, three times at monthly intervals and bled 15 days after the last injection. C3H and Swiss mice were immunized i.p. with 50 ,ug OA adsorbed to 5 mg A1(OH)3 gel and bled after 14 days. The short-term immunization permitted the investigation of the effect of small tumours on active systemic anaphylaxis.
Passive cutaneous anaphylactic reactions (PCA) The dorsal skin of mice was shaved and 30 ,l of serial dilutions of antiserum injected i.d. After either 2 h or 48 h (i.e. the latent period, used to detect IgGl or IgE activity respectively) 100 ,ug of OA was injected i.v. in 0 2 ml of 0 5 per cent Evans blue dye. The intensities of the PCA reactions were scored on an arbitrary 0-4 scale and the PCA titre was defined as the reciprocal of the serum dilution giving threshold reactions in at least two of three recipients. PCA reactions in McC3-bearing C3H mice were performed in the dorsal skin, while the tumour was always grafted ventrally. Haemoconcentration The technique utilized was basically that of Iff & Vaz (1966). Small volumes of blood (5 ,ul) were taken from mice by bleeding from the orbital plexus before anaphylactic challenge, then 5 and 10 min after. The samples were diluted in 2 5 ml of 1 per cent Na2CO3 and the optical density at 541 nm read. The severity of hypovolaemic shock was assessed by the percentage increase in blood haemoglobulin concentration after challenge. Challenges with histamine and serotonin mixtures were also performed 4 days after the i.p. injection of 1010 merthiolate-killed B. pertussis organisms (J. Raynaud Institute Pasteur). This treatment greatly increases the sensitivity of normal animals to the action of vasoactive amines (Munoz & Bergman, 1968). Histamine assay Serum histaminase levels were assayed by the technique of Roscoe & Kupfer (1972) as modified by Lin, Orcutt & Stolbach (1975) where, after incubation of 14C-labelled histamine with the serum, the deaminated reaction products were separated from the unreacted parent molecule on phosphocellulose and then quantified by ,B counting. Also, to test the anti-tumour activity of histaminase inhibitors, C3H mice were treated with aminoguanidine hemi-sulphate (Sigma) in their drinking water at a level of 20 ug/ml for a week before
Immediate hypersensitivity reactions in mice grafting with McC3 and then for the following 6 weeks. Tumour diameters were measured twice weekly. RESULTS Anaphylactic challenge of C3H mice bearing McC3 tumours
Normal or McC3 tumour-bearing (10-20 mm diameter) C3H mice were anaphylactically challenged by the means described below, and the severity of hypovolaemic shock assessed by quantifying the resulting haemoconcentration. In general, animals with larger McC3 tumours (> 20 mm) could not be used because these mice were severely anaemic, with very low starting haemoglobin concentrations. Inconsistent results were obtained with such animals. Active systemic anaphylaxis (ASA) was induced by sensitization with OA adsorbed to A1(OH)3 gel and i.v. challenge with OA after 14 days. The animals with McC3 tumours had received grafts 2 days after sensitization and the anti-OA PCA titres in a pool of these day 14 sera were 320 for IgG1 and 80 for IgE, compared with 320 and 160 respectively in the control. The data presented in Table 1 demonstrate that the severity of ASA was significantly lower in McC3 tumour-bearing C3H mice than normal. The intensity of passive systemic anaphylaxis
647
(PSA) mediated by a DBA/1 anti-OA antiserum was also significantly lower in C3H mice carrying the McC3 tumour (Table 1). C3H mice with McC3 tumours were more resistant than normal to challenge with a mixture of the amines primarily responsible for anaphylactic shock, histamine and serotonin (Table 1). However, both normal and tumour-bearing C3H mice were sensitized to the vasoactivity of these amines by treatment with B. pertussis vaccine (Table 1). Histaminase levels were estimated in the serum of normal and tumour-bearing C3H mice but were bearly detectable, being < 0 05 ,g amine hydrolyzed/ ml/h, in both situations. Also, McC3 tumour grafts grew no less rapidly than usual in C3H mice receiving the histaminase inhibitor aminoguanidine in their drinking water. PCA titres in C3H mice bearing McC3 tumours The 2 h (IgG1) and 48 h (IgE) PCA titres of anti-OA antisera were determined in either normal C3H mice or C3H mice bearing McC3 tumours. Despite the dark pigmentation of the outer layer of C3H mice skin, the PCA reactions were well defined when the skin was reflected and the clear inner skin layer examined. The PCA titres were easily determined in normal C3H mice. In C3H mice carrying large (15-25 mm diameter) McC3 tumours the PCA reactions were diffusely ill-defined and the titres
Table 1. Anaphylactic challenge of C3H mice bearing McC3 tumours
Recipient*
Normal C3H C3H with McC3
Mean per cent haemoconcentration
ASAt
PSAt
H+ S§
B. pertussis; H+ S¶
26+4 P
