31

Immune Functions¹

Henry J. Winn

Immunology in a broad sense encompasses a wide range of investigations bearing on the complex problems of resistance and susceptibility of higher forms of life to infectious diseases, and it draws heavily on a variety of medical, biological, and chemical disciplines. Experimental immunology has, however, focused largely on the antibody response, which in mammals tends to play a pivotal role in acquired immunity. This preoccupation with antibody, originally inspired by the dramatic demonstrations of its association with specifically increased resistance to some diseases and subsequently attributable to an increasing awareness of the practical and conceptual implications of specific immune reactions for many areas of biology, has nurtured the development of a conception of immunology that is much narrower in scope than originally proposed. It is immunology in this restricted sense that is to be considered here. The basic principles of the field are presented in detail in a number of texts ( Boyd, 1956; Raffel, 1961; Humphrey and White, 1963). The present discussion will be by no means exhaustive, but is intended to direct interested readers to published studies which provide basic information on immune functions in mice or in which some unique biological trait of these animals has been exploited in immunological investigations.

ANTIBODIES

Antibodies are serum globulins that are capable of combining specifically with substances (antigens) which provoke their formation by cells of the lymphoid tissues. They belong to a class of plasma proteins, designated γ-globulins or immunoglobulins, which shows a high degree of physical and chemical heterogeneity. Indeed the term γ-globulin may be somewhat misleading since antibody activity may be found in the β-globulins or α₂-globulins. However, despite this lack of true homogeneity, there is much justification for treating antibodies and similar proteins elaborated by plasmacytes and lymphoid cells as a family of closely related proteins under a single inclusive designation. In addition to their common tissue origin they may have many overlapping physical and chemical properties and they have strikingly similar immunochemical characteristics (review by Fahey, 1962). The term immunoglobulins will be used here but it must not be interpreted to mean that all of the protein molecules so designated have known antibody activity, and the use of a collective designation should not obscure the fact that relatively small differences in molecular structure of antibodies can be responsible for marked differences in the biological activities of the molecules.

It has been possible to establish defined subfractions of the immunoglobulins and with respect to the mouse Fahey et al. ( 1964a) have described four major subdivisions. Two classes of 7 S γ-globulins differing in electrophoretic mobility and in antigenic structure were designated 7 S γ₁-globulins and 7 S γ₂-globulins. A third fraction designated γ_1A-globulin is found chiefly in the β-globulin fraction. It is heterogeneous with respect to molecular size and antigenically distinct from the other immunoglobulins. The fourth class, γ₁-globulin, is a high molecular weight fraction which migrates on electrophoresis with the mid γ-globulins, and contains characteristic antigenic determinants. An additional component, designated X_γ, was also demonstrated in the γ-globulins of mice but was considered to be devoid of immunological function and, accordingly, was not classified as an immunoglobulin. Each of the four major classes was shown to contain antibody activity. In a subsequent study Fahey et al. ( 1964b) described two subclasses of mouse 7 S γ₂-globulins which were designated γ_2A-globulins and γ_2B-globulins. Enzymatically produced subunits of mouse immunoglobulins have been studied by Fahey and Askonis ( 1962a, 1962b). Classification of immunoglobulins on the basis of genetically determined isoantigens ("alloantigens," Chapter 24) is discussed in Chapter 8.

Purified antibodies obtained from the serum of mice immunized with protein conjugates were fractionated and some biological properties of the fractions were studied by Nussenzweig et al. ( 1964). Two types of precipitating antibody were found and designated γ₁ and γ₂. Both types were found in the γ-region on electrophoresis in agar gels, the γ₁ migrating slightly more rapidly than the γ₂. Mouse γ₁ antibodies were capable of sensitizing mice but not guinea pigs for passive cutaneous anaphylaxis, but they did not cause detectable lysis, in the presence of mouse complement, of sheep erythrocytes coated with the protein conjugates used as antigens. The γ₂-antibodies, in conjunction with mouse complement, lysed the coated red blood cells and were capable of transferring passive cutaneous anaphylaxis to guinea pigs but not to mice. These interesting differences may be related to the differences between mouse and guinea pig complement systems which are described in the next section.

The kinds and amounts of immunoglobulins elaborated in response to antigenic stimuli may be influenced by: (1) the chemical and physical properties of the antigen and the vehicle in which it is dissolved or suspended, (2) the route of injection, (3) the intensity and duration of immunization, (4) the age of the recipient animal, and (5) genetic factors. All of these factors interact and the roles they play in directing the course of immune responses are poorly understood.

Fahey and Humphrey ( 1962) studied the antibody responses of C3H/He mice to single intravenous injections of pneumococcal polysaccharide, hemocyanin, or sheep erythrocytes. Antisera were examined 6 days after the administration of antigen. Antibodies to both of the soluble antigens had the properties of low molecular weight (6.5 S) γ-globulins, whereas the bulk of the anti-erythrocyte antibodies were γ-macroglobulins (19S) with a small proportion found among the lower molecular weight globulins. Subsequently, Fahey and Lawrence ( 1962) compared the physicochemical and immunological properties of antibodies that had been produced against hemocyanin in eight inbred strains of mice. Most of the antibodies from all of the mice were 6.6 S γ-globulins but there were marked differences in the electrophoretic properties of the antibodies from different strains. Winn ( 1965a) found that the response of a large number of inbred strains of mice to a single injection of sheep erythrocytes was confined almost exclusively to the elaboration of higher molecular weight antibodies. However, after hyperimmunization there were marked differences in the responses of various strains. C57BL/10J mice continued to produce large amounts of antibodies of the macroglobulin type with barely detectable amounts of low molecular weight antibodies. In A/J mice the production of high molecular weight antibodies was no longer detected, though their sera contained high titers of low molecular weight antibodies. Mice of other strains produced mixtures of the two types of antibodies in various proportions. The importance of genetic factors is also indicated in a study showing quantitative differences in the immune response to tetanus toxoid between four inbred lines of mice ( Ipsen, 1959).

The immune responses of very young animals have been found to differ appreciably from those of mature animals. This aspect of immunization has not been investigated intensively in mice, but Boraker and Hildemann ( 1965) have examined the sera of young A/Jax and C57BL/6J mice that had received reciprocal grafts of skin and lymphoid tissues. They found that antibodies detected prior to 20 and 27 days of age in C57BL/6J and A/Jax mice, respectively, were of the macroglobulin type. Older animals of either strain produced 7S antibodies as well as macroglobulins.

The interactions of mouse antibodies with specific antigens become manifest in a variety of ways that are characteristic of similar interactions of antibodies from other species. Most of these manifestations are alluded to in various parts of this discussion and require no further comment. It is appropriate, however, to mention the study of Anacker and Munoz ( 1961) on the precipitin reaction involving ovalbumin and the corresponding antibody contained in mouse peritoneal fluid. Mice repeatedly injected by the intraperitoneal route with antigen in Freund's adjuvent developed large volumes of ascites fluid containing high concentrations of antibody, The antibody was qualitatively indistinguishable from that found in serum and behaved very similarly to rabbit antibody in quantitative precipitin tests. Insoluble precipitates were found throughout the region of antibody excess and soluble complexes were found in the region of antigen excess. The amounts of antibody precipitated at equivalence reached, in some cases, the unusually high level of 2 mg of protein nitrogen per milliliter.

COMPLEMENT

Although it is widely recognized that complement may play an important role in many diverse immunological reactions, the detection and assay of this complex group of plasma proteins are generally based on its ability to lyse sheep erythrocytes sensitized with rabbit anti-sheep cell antibodies. Measurements based on this assay system have invariably revealed low or undetectable levels of activity in mouse sera. This has been attributed to deficiencies in various individual components of complement ( Borsos and Cooper, 1961; Herzenberg et al., 1963). Borsos and Cooper showed that pools of serum from CFW mice contain all of the recognized factors of the complement system, that the C'2 and C'3 activities are quite low, and that mouse serum contains a potent inhibitor of lytic activity. These findings provide a suitable explanation for the failures of a number of workers to detect complement activity in mice. However, Herzenberg et al. reported that a deficiency of hemolytic complement in a highly inbred strain of mice was genetically determined. This deficiency was shown to be controlled by a single genetic locus (Hc) and subsequent studies have revealed a similar or identical deficiency in a number of inbred lines.

Erickson et al. ( 1963) showed that genes at this locus controlled the production of a serum protein identified by immunochemical techniques. Their evidence suggests that the allele carried by mice of deficient strains has no recognizable product. Subsequent studies by Terry et al. ( 1964) have suggested that deficient strains lack one of the components designated collectively as C'3. There is no indication that the health or immune functions of the deficient mice are impaired.

The hemolytic activity of mouse complement has been shown to be more effectively activated by 7S hemolysins than by high molecular weight antibodies ( Winn, 1965a). This is in striking contrast to the activation of guinea pig complement which has served as a prototype in this area of research. Winn showed that this difference between complement from mice and guinea pigs could be attributed to differences in the C'1 macromolecules of the two species. Additional data presented in this report show that in hemolytic systems involving 19 S hemolysins, mouse serum markedly inhibits the activity of guinea pig complement but supplements that of rabbit serum. The inhibitory properties of mouse serum lead to some difficulties when mouse antisera are employed in complement-fixation tests. Problems of this nature are discussed by Winn ( 1965b), who has suggested ways of eliminating or compensating for this type of interference.

HYPERSENSITIVITY

The development of the immune state is often accompanied by a dramatic increase in reactivity to further contact with the same antigen. This increased sensitivity, referred to as allergy or hypersensitivity, has divers manifestations which lead to tissue destruction varying in degree of severity from evanescent wheal and flare reactions to chronic inflammation which may proceed to intense suppuration and necrosis. In some case severe generalized illness or death of a sensitive animal may occur within a few minutes after contact with antigen. The substances responsible for the development and expression of hypersensitivity are of widely varying chemical constitution, and they may or may not have intrinsic toxic properties. Microorganisms (dead as well as living), microbial metabolites, foreign proteins, pollen extracts, and some low molecular weight organic compounds provide only a sample of the known allergens.

Hypersensitive reactions are conveniently divided into immediate and delayed types on the basis of the temporal sequence of reactions following contact with antigen. Immediate-type reactions can be detected within seconds after contact and are often, though not necessarily, relatively short-lived. Delayed-type reactions are detectable by ordinary means only several hours after application of the incitant, and they continue to develop in intensity for periods of 18 to 72 hours. These differences in the tempo of response reflect basic differences in the mechanisms responsible for the development of the reactions. Reactions of the immediate type are invariably associated with serum antibodies, whereas delayed-type reactions involve cell-bound or cell-associated antibodies. A clear distinction between the two types is provided by demonstrations that a single antigen may induce either type of reactivity alone. Subdivision of the allergic reactions, particularly those of the immediate type, is useful and will be indicated below in discussing these reactions in mice.

The occurrence of delayed-type responses in mice has been reported (review by Crowle, 1959a), but they are not elicited so regularly as in other species and typical cutaneous reactions are rarely observed. Apparently, mice have the capacity to elaborate the immune substances that mediate delayed hypersensitive responses, but they seem to be constitutionally incapable of displaying reactions of the type observed in other animals. By employing a variety of special techniques, Crowle ( 1959b) has been able to demonstrate specifically inducing inflammatory responses that are evidently manifestations of delayed reactivity.

Additional information on this point comes from studies on experimental allergic encephalomyelitis. There is a good deal of evidence that the production of this condition depends on the establishment of delayed-type reactivity to brain tissue. The ability to produce the disease regularly in mice supports the conclusion that these animals can develop reactivity of the delayed type ( Lee and Olitsky, 1955).

Immediate-type allergy may be divided into two classes. Anaphylactic reactions result from the rapid release of short-lived pharmacologically active agents immediately following interaction of antigen and antibody in vivo. The symptoms and severity of these reactions vary considerably in the different species principally because of differences in the amounts and kinds of active substances released and in the sensitivities of various animals to the action of these compounds. Arthus reactions are local responses initiated directly by the formation of antigen-antibody precipitates. Apparently, these precipitates have an irritant effect on the walls of small blood vessels, leading to the development of intense inflammatory responses. The reaction begins very shortly after application of a test antigen and continues to develop in intensity for several hours.

With respect to systematic anaphylaxis, older views that mice are highly resistant to the induction of allergic reactivity have been completely reversed. Burdon ( 1937) and Weiser et al. ( 1941) showed conclusively that mice can be sensitized and shocked, though the course of the reaction is not nearly so violent as that observed in other species. Quite commonly death does not occur until several hours after the challenge. During this interval the mice usually remain quiet and move only if disturbed. The fur is ruffled, there may be scratching of the eyes and muzzle, and there may be some evidence of respiratory difficulty. As death approaches, breathing becomes more difficult, cyanosis may be marked, and the animals display evidence of paralysis or convulsive kicking. Occasionally, death occurs as soon as 15 minutes after injection of antigen.

Cameron ( 1956) established arbitrary classes of nonfatal shock on the basis of the severity and duration of symptoms in W-series mice. His report includes a study of the relative sensitizing abilities of various proteins, the efficacy of various routes for sensitizing and challenging, the effect of varying the number of sensitizing doses, and the duration of sensitization. Quantitative estimation of anaphylactic sensitivity in mice has been based on observations that shock is accompanied by a fall in body temperature ( Kind, 1955) and a rise in hematocrit ( Harris and Fulton, 1958). McMaster and Kruse ( 1949) have devised an extremely sensitive technique based on the detection of vasospasm in the ears of intravenously injected mice that had been previously sensitized with minute quantities of antigen.

Sobey and Adams ( 1959) have demonstrated in mice a high heritability of anaphylactic sensitivity to bovine plasma albumin, and Fink and Rothlauf ( 1954) reported variations in sensitivity to shock in five inbred strains of mice injected with ovalbumin. It is not clear whether these variations are attributable primarily to differences in the kinds and amounts of antibody formed in response to these antigens or to differences in sensitivity to the pharmacologically active compounds released as a result of the combination of antigen and antibody.

Pharmacological aspects of anaphylaxis in mice have been discussed by Austen and Humphrey ( 1963). The subject is too complex and controversial to treat here in any detail, but much experimental data suggest that histamine does not play an important role, and there is some evidence implicating serotonin as a mediator of shock.

A peculiar feature of systematic anaphylaxis in mice is brought to light by studies carried out in animals that have received injections of Hemophilus pertussis vaccine (review by Kind, 1958). This treatment often leads to hyperreactivity to histamine. When vaccine is administered simultaneously with a sensitizing antigen, the subsequent challenge with antigen results in a much higher mortality than in mice sensitized with antigen alone. The mechanism of this potentiating effect is not understood, but there seems to be no clear-cut relationship between the increased sensitivity to shock and the enhanced reactivity to histamine. Marked differences in the reactions of various inbred strains of mice are apparent but no comprehensive studies have been reported.

The combination of small amounts of antigen and antibody in cutaneous sites leads to an increased permeability of small vessels permitting local concentration of intravenously injected dyes. The reaction has been used principally for the detection of minute quantities of specific antigens or antibodies, though some characterization of the biological activities of different classes of antibodies may also be achieved. This cutaneous form of anaphylaxis has been described in the mouse by Ovary ( 1958) and immunochemical aspects of such reactions were reported by Nussenzweig et al. ( 1964).

A great deal of our information on the mechanism of anaphylaxis has come from studies carried out in vitro with isolated tissues from actively or passively sensitized animals. Experiments have been carried out with a variety of tissues from different animals (review by Austen and Humphrey, 1963), but the use of mice has been restricted to the study of isolated strips of uterine muscle ( Fink and Rothlauf, 1955). Tissue placed in an organ bath with nutrient solution showed characteristic contractions after addition of the antigen to which the mice had been sensitized. The procedure is often referred to as the Schultz-Dale technique in recognition of the workers who first demonstrated the phenomenon with guinea pig tissue. These experiments with mouse tissue support the suggestion that serotonin rather than histamine plays an important role in anaphylaxis in mice.

Typical Arthus reactions are difficult to produce in mice and the inflammatory responses accompanying these reactions are not so intense as those observed in rabbits. Reactions have been elicited in the foot pads of immunized mice but the lip is a more convenient site of injection. Moreover, Arthus reactions are more regularly obtained by intralabial injection and in this area they are more readily distinguished from nonspecific inflammatory responses ( Freund and Stone, 1956).

TRANSPLANTATION IMMUNOLOGY

The biology of tissue transplantation has been reviewed comprehensively by Russell and Monaco ( 1964) and many aspects of the problems of transplantation as they relate to mice are discussed in Chapter 24. The conception of homograft rejection as a manifestation of the immunization of the host against isoantigens of the graft is universally accepted, though the nature of the immune substances responsible for destruction of grafted tissues is not known precisely. There are striking differences in the sensitivity of mouse cells derived from various tissues to the destructive effects of isoimmune sera and complement (reviews by Winn, 1962; Stetson, 1963; Möller, 1963), and in some cases it has been shown that the presence of serum antibodies in the host may lead to prolonged survival of homografts ( Kaliss, 1962; Snell et al., 1960). A substantial body of data indicates the participation of cellular or cell-bound antibodies in graft destruction (reviewed by Snell, 1963). Comprehensive reviews of the relative importance of humoral and cellular factors in graft rejection have been published by Gorer ( 1961), Brent ( 1958), and Amos ( 1962).

SUMMARY

This discussion has dealt briefly with a description of the kinds of immune substances that have been found in mice and with some aspects of their biological activities. The points which serve to distinguish immunological reactivities of mice from those of other species have been emphasized, and differences in reactivity between inbred strains of mice have been described. Important areas of immunology have been omitted because little definitive information with respect to mice is available or because the subject is such that no special treatment is required beyond that found in textbooks on general immunology.

¹The writing of this chapter was supported in part by Public Health Service Research Grant CA 01329 from the National Cancer Institute.

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