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Hereditary Immunodeficiencies This chapter describes the genetics, pathophysiology, husbandry, and re- production of 64 inbred, hybrid, and mutant strains of rodents with hereditary immunodeficiencies. It is not an exhaustive review, but rather, it summarizes current knowledge and provides key references as a starting point for the reader. Most of the animals described do not need extraordinary methods of isolation and maintenance, but do require procedures that prevent the intro- duction of rodent pathogens. The procedures necessary for the care of those animals susceptible to ubiquitous organisms are detailed in Chapter 4. Strains to be discussed are listed in Table 2-1. MICE WITH SINGLE MUTATIONS Beg:, Begs (Resistance and Susceptibility to Mycobacterium bovis); Ityr, Ids (Resistance and Susceptibility to Salmonella typhimurium); [star, [shS (Resistance and Susceptibility to L`eishmania Donovan Genetics Resistance and susceptibility alleles of Beg, Itcy, and Lsh define relative resistance to in vivo infection with Mycobacterium bovis (Gros et al., 1981) or M. Iepraemurium (Skamene et al., 1984; Brown and Glynn, 1987), Sal- monella t~yphimurium (Plant and Glynn, 1979), and Leishmania donovani (Bradley et al., 1979), respectively. Phenotyping of several inbred, recom 36

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HEREDITARY IMMUNODEFICIENCIES 37 TABLE 2-1 List of Immunodeficient Rodents Dysfunctions Other than Care Immunodeficiency Special Special Page Auto- Non- Breeding Husbandry Mutation or Strain No. immunity immune Techniques Procedures Mice with single mutations Bcgr; Bcgs (resistance or susceptibility to Mycobacterium bovis) 36 - - - - bg (beige) 40 - + db (diabetes) 43 + + + + df (Ames dwarf) 46 - + Dh (dominant hemimelia) 47 - + + dw (dwarf) 49 - + + + gl (gray-lethal) 51 - + + gld (generalized lympho proliferative disease) 52 + Hc (hemolytic complement) 54 hr (hairless); hrrh (rhino) 55 - + + Ityr; ItyS (resistance or susceptibility to Salmonella typhimurium) 36 Ih (lethargic) 58 - + + Ipr (lymphoproliferation) 59 + - + Lps ~ ( lip op olyac charide response, defective) 62 Lshr; Lshs (resistance or susceptibility to Leishmania donovani) 36 me (motheaten); meV (viable motheaten) 64 + + mi (microphthalmia) 67 - + + + nu (nude); nuS'r (streaker) 69 + + + + ob (obese) 72 + + + oc (osteosclerotic) 73 - + + + op (osteopetrosis) 75 - + + + scid (severe combined immunodeficiency) 77 - - - + Tol-1 (tolerance to bovine gamma-globulin) 80 Continued

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38 IMMUNODEFICIENT RODENTS TABLE 2-1 (continue~ Dysfunctions Other than Care Immunodeficiency Mutation or Strain Page Auto- Non No. immunity immune Special Breeding Techniques Special Husbandry Procedures Mice with single mutations (continued) vit (vitilago) 80 W (dominant spotting); wv (viable dominant spotting) wst (wasted) xid (X-linked immune deficiency) Yaa (Y-linked autoimmune accelerator) S-region-linked genes controlling murine C4 Mice with multiple mutations gld xid Ipr nu Ipr xid Ipr Yaa nu bg nu xid nu bg xid nu Dh Yaa bg Yaa xid Inbred mice BSVR, BSVS BXSB/Mp (females) DBA/2Ha MRL/Mp NOD NON NZB NZB x NZW F~ (BWFi) NZB x SWRF~or SWR x NZB F~, both abbreviated SNF~ PN SAM-P SJL/J SL/Ni / 108 112 82 84 - + 85 87 89 90 90 91 91 91 92 92 93 93 94 94 96 98 98 100 + 103 104 + + + + + + + _ + _ - + + + 1 + + + + + + _ _ + + + + + + + + _ _ + - + + - _ + +

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HEREDITARY IMMUNODEFICIENCIES 39 TABLE 2-1 (continued) Dysfunctions Other than Immunodeficiency Page Auto- Non Mutation or Strain No. immunity immune Care Special Breeding Techniques Special Husbandry Procedures Outbred mice SWAN Rat mutants ia (incisor absent) op (osteopetrosis) rnu (Rowett nude); rnuN (n nu, New Zealand nude) It (toothless) C4 deficiency Inbred rats BB/Wor LOU/C Guinea pig mutants C2 deficiency C3 deficiency C4 deficiency Hamster mutants nu C6 deficiency 122 123 124 125 128 129 130 134 134 135 136 138 139 + + + _ + + + + NOTE: A discussion of each positive ( + ) listing follows in the narrative describing each specific mutant or strain. binant inbred, and congenic strains indicates that the resistance and susceptibility alleles for all of these unrelated, obligate, intracellular pathogens are identical or closely linked on chromosome 1 (Plant et al., 19821. The BALB/c, C57BL/6, C57BL/lOScSn, and DBA/1 strains carry the susceptibility al- lelefs); the A/J, C3H/HeJ, C57L, and DBA/2 strains have the resistance allelefs). BALB/c (Potter et al., 1983) and C57BL/lOScSn (Blackwell, 1985) congenic strains carrying the resistance allelefs) have been developed using DBA/2 and C57L, respectively, as donor strains. Although these loci do not affect the immune response per se, they are unique in affecting macrophage function. There are other loci that affect resistance to Mycobacterium bovis, M. Iepraemurium, Salmonella typhi- murium, and Leishmania donovani; however, these have not yet been mapped (Curtis et al., 1982; Curtis and Turk, 19841.

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40 IMMUNODEFICIENT RODENTS Pathophysiology Short-term culture of splenic and hepatic cells indicates that the phenotype is expressed in macrophages, which vary in their ability to control the rate of intracellular replication of pathogens (Lissner et al., 1983~. Denis et al. (1988) evaluated the efficiency of mouse splenic macrophages from strains congeneic for the Bcg locus and found that macrophages from Bcgr mice were superior to those from Bcgs mice in presenting bacteria-derived soluble and particulate antigens. In addition to imparting resistance to infection to Salmonella typhimurium and Leishmania donovani, the Bcg locus plays a less significant role in the protection of the closely related agents Salmonella typhi and Leishmania major. In the case of L. major, Davies et al. (1988) found that the parasite has developed a sophisticated method for escaping the effects of the Bcg gene. It induces increased monocyte infiltration into the site of infection, thereby providing safe targets in which the parasite can survive and multiply. Husbandry Special husbandry procedures are not required. Reproduction These animals reproduce normally. bg (Beige) Genetics Beige (formerly also called slate) is a recessive mutation located on chro- mosome 13 that arose independently several times. The symbol bg was assigned to a mutation, probably radiation induced, found at Oak Ridge National Laboratory, Oak Ridge, Tenn. (Kelly, 19571. A spontaneous mu- tation called slate (sit) was discovered at Brown University in 1955 (Chase, 1959) and was first described in 1963 (Pierro and Chase, 19631. In 1965 sit was recognized to be allelic with bg, and Chase (1965) recommended that the symbol sit be dropped if the mutant proved to be identical to bg. Another mutant allele, Egg, occurred spontaneously in the C57BL/6J strain at the Jackson Laboratory, Bar Harbor, Maine (Lane, 1962~. Beige-2J (bg2~) arose spontaneously in strain C3H/HeJ at the Jackson Laboratory in 1972.

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HEREDITARY IMMUNODEFICIENCIES 41 A C57BL/6J-bg/bg mouse (rear) and a C57BL/6J control (front). Mice homozygous for the beige mutation show a dilution in pigmentation. Photograph courtesy of the Jackson Laboratory, Bar Harbor, Maine. Pa thop hys to logy Homozygous beige mice have a light coat color and reduced ear and tail pigmentation. Eye color is light at birth, changing to a color that varies from ruby to almost black in adults. The phenotypic manifestations in bgibg mice closely resemble the Chediak-Higashi syndrome in humans, the Aleutian trait in mink, and a syndrome of partially albino Hereford cows (Lutzner et al., 1967). Beige homozygotes have been reported to have cytotoxic T-cell and mac- rophage defects (Mahoney et al., 1980; Saxena et al., 1982; Halle-Pannenko and Bruley-Rosset, 1985~. They have abnormally large lysosomal granules in a wide variety of cells, including leukocytes of bone marrow and peripheral blood, thyroid follicular cells, type II pneumocytes, mast cells, pyramidal cells and pericytes of the cerebral cortex, Purkinje cells of the cerebellum, spinal cord neurons, islet and acinar cells of the pancreas, liver parenchymal cells, and proximal tubule cells of the kidneys (Oliver and Essner, 1973; Chi et al., 1978; Prueitt et al., 19781. The defective granules are thought to contribute to lowered chemotaxis and a general motility defect (Gallin et al., 19741. Peritoneal exudate leukocytes of C57BL/6J-bg/bg mice contain very low or undetectable levels of neutral protease (Vassalli et al., 1978), and cir- culating polymorphonuclear leukocytes (PMNs) in bg homozygotes have

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42 IMMUNODEFICIENT RODENTS impaired chemotactic and bactericidal activities. Kaplan et al. (1978) showed that the addition of small numbers of normal platelets or serotonin to whole blood of beige homozygotes increased the bactericidal activity of the PMNs. They theorized that normal platelets or serotonin enhanced the formation of cellular cGMP, thereby promoting polymerization of tubulin to microtubules and reversing abnormal bactericidal activity. A selective impairment of NK-cell function in bgibg mice prevents the initiation of antibody-dependent or antibody-independent cytolysis of tumor cells. The exact nature of the defect is not known, but it is believed to lie in the cell's lyric mechanism (Roder and Duwe, 19791. In vitro, these NK cells can be activated by treatment with interferon (Brunda et al., 19801. Defects have also been reported in cytotoxic T cells and macrophages (Ma- honey et al., 1980; Saxena et al., 1982; Halle-Pannenko and Bruley-Rosset, 19851. Beige homozygotes show a greater susceptibility to infection with pyogenic bacteria than do normal controls. This appears to be due to a defect in lysosomal function. The impairment is not of humoral origin because serum immunoglobulin levels are comparable in homozygotes, heterozygotes, and normal controls (Elin et al., 19744. Lane and Murphy (1972) demonstrated that the bg gene confers a greater susceptibility to spontaneous pneumonitis. In their study with SB/Le mice, which are homozygous for bg, sa (satin), and Aw (white-bellied agouti), and backcross offspring from an outcross to C57BL/6J-AW-~, beige homozygotes (sa bgisa bg and + bgisa bg) had a significantly higher incidence of spontaneous pneumonitis than did nonbeige mice (sa +Isa bg and + +Isa by). C57BL/6J-bg'/bg~ and C3H/HeJ- bg2'lbg2' mice do not develop spontaneous pneumonitis under similar ex- perimental conditions (J. B. Roths, The Jackson Laboratory, Bar Harbor, Maine, unpublished data). C57BL/6 mice carrying the bg mutation or the pigment mutations pallid (pa), pearl (pe), light ear (le), pale ears (ep), maroon (ru-2mr), or ruby eye (ru) have hypopigmentation, prolonged bleeding time, normal platelet num- bers accompanied by reduced platelet granules, and decreased levels of plate- let serotonin. These seven mutations map to separate chromosomal sites; however, bone marrow transplantion from normal C57BL/6 mice to irradiated mutants corrects the defects in platelet serotonin and bleeding time. This suggests that in each case there is a cellular basis to the deficiency (Novak et al., 1985; McGarry et al., 19861. Experimentally, beige mice have played an important role in studies of hematopoietic differentiation. The giant lysosomal granules of beige mice provide an exquisite cytoplasmic marker for mast cells, PMNs, and osteo- clasts in bone marrow chimeras (Murphy et al., 1973; Ash et al., 1980; Kitamura et al., 19811. C3H/HeJ-bg27/bg9~ mice that survive to 17 months of age show a pro

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HEREDITARY IMMUNODEFICIENCIES 43 gressive neurological disorder accompanied by a nearly complete loss of cerebellar Purkinje cells (Murphy and Roths, 1978b). This lesion is less severe in similarly aged C57BL/6J-bgi/bg~ mice. The pigment mutations ep, pa, and rp (reduced pigmentation) affect ly- sosomal functions and lead to suppressed NK cell activity (Orn et al., 1982~. An additional 29 pigment mutations have been described in mice, all of which are known to affect lysosomal biogenesis. However, little is known about the function of the immune system in these mice (Brands et al., 198 1~. Husbandry Beige mice are more susceptible than immunocompetent mice to challenge by a wide variety of infectious agents, but they do not suffer from infections caused by indigenous microorganisms that are not pathogenic to immuno- competent mice. These animals survive well in a pathogen-free environment. Barrier isolation, as described in Chapter 4, is not required for the mainte- nance of these animals. Reproduction Beige mice of both sexes will breed. db (Diabetes) Genetics The mutation diabetes (db) on chromosome 4 is a spontaneous autosomal recessive obesity gene discovered at the Jackson Laboratory, Bar Harbor, Maine, in the C57BL/KsJ inbred strain (Hummer et al., 19661. The gene has been transferred to the C57BL/6J and a number of other inbred strains. Several other alleles are present at the db locus: dab arose in an inbred brown (b) whirler (wi) stock (Lane, 1968), db3~ arose in strain 129/J (Letter et al., 1980), and dbPaS arose in strain DW/J (Aubert et al., 19851. Another allele, dba4, arose in a stock selected for large size and was called adipose (ad) (Falconer and Isaacson, 19591. In 1972 the gene was found to be allelic with db and was redesignated dboa' (Hummer et al., 19731. Pathophysiology Regardless of inbred strain background, dbidb mice always exhibit a marked obesity associated with hyperphagia, hyperinsulinemia, and severe insulin resistance. However, ultimate development in dbidb mice of a permanent diabetic condition (typified by chronic hyperglycemia, reduced serum insulin

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44 IMMUNODEFICIENT RODENTS A 129/J-db3J/db3J mouse (left) and its normal littermate (right). Mice homozygous for the diabetes mutation develop non-insulin-dependent diabetes and become obese. Photograph cour- tesy of the Jackson Laboratory, Bar Harbor, Maine. levels, beta-cell necrosis, and pancreatic islet atrophy) depends entirely on the inbred strain background and gender (Coleman, 1 978; Leiter et al., 1 98 1 ). The mutation was named diabetes because the C57BL/KsJ strain of origin proved susceptible to its diabetogenic action. In this strain, dbldb mice of both sexes develop an early-onset diabetes resembling in some respects the human non-insulin-dependent (type II) diabetes mellitus. On the contrary, the same mutation on the diabetes-resistant C57BL/6J inbred background did not produce beta-cell necrosis or permanent hyperglycemia; instead, the dia- betogenic action of the mutation was well compensated by unrestricted hy- perplasia of pancreatic beta cells and sustained hyperinsulinemia (Coleman, 19789. Although type II diabetes in humans is not generally associated with an autoimmune etiology, the reports of both humoral and cell-mediated autoim- munity against pancreatic beta cells in C57BL/KsJ-db/db mice (Debray-Sachs et al., 1983), as well as the finding of immune complex deposition in the kidneys (Meade et al., 1981), suggested that this model might be an amalgam of features of type I (autoimmune) as well as type II diabetes in humans. An association between the H-2 haplotype and diabetes susceptibility and resistance was initially suggested by a comparison of db gene expression on a variety of inbred strain backgrounds (Letter et al., 1981~. However, seg- regation analyses have shown that H-2 does not control susceptibility; instead, male gender-linked factors, and possibly endogenous retroviral genes, appear to be the major modifiers of diabetes severity (Letter, 1985; Leiter et al., 1987a). The question of the pathogenic role of the humoral and cell-mediated anti-beta-cell reactivities in dbldb mice has also been resolved by combining -

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HEREDITARY IMMUNODEFICIENCIES 45 this mutation with immunodeficiency genes such as severe combined im- munodeficiency (scid) to compromise T- and B-lymphocyte function (Letter et al., 1987b). This study showed that immunodeficient dbidb mice still developed diabetes, indicating that autoimmunity is probably a reflection of islet cell destruction rather than its cause. The C57BL/KsJ-db/db mouse has proven to be exceptionally useful as a model for analyzing the effects of chronic non-insulin-dependent diabetes on the immune system. There is early thymic involution and T lymphopenia from 8 weeks onward (Boillot et al., 19861; the disturbed metabolic milieu leads to depressed cell-mediated immunity and lymphokine production (Fer- nandes et al., 1978; Mandel and Mahmoud, 1978; Kazura et al., 1979; Pasko et al., 19811. Many of the T-cell functions that are impaired in vivo (for example, generation of alloreactive cells) are normal when assessed in vitro (Fernandes et al., 1978), which demonstrates the suppressive effect of the imbalanced metabolic environment in vivo. In contrast to the suppression of T-lymphocyte functions, certain B-lymphocyte functions are increased, in- cluding autoantibody production against islet cell cytoplasmic antigens, thymic hormones, and insulin (Dardenne et al., 1984; Serreze et al., 1988b; Yoon et al., 1988~. Transfer of dbldb marrow cells into lethally irradiated +/+ recipients rescued recipient mice from radiation death but did not transfer the diabetes syndrome (Letter et al., 1987b). Husbandry Special procedures are not required to maintain these animals; however, their life span can be prolonged by dietary restriction, and the severity of the syndrome can be significantly diminished by feeding carbohydrate-free, protein-enriched defined diets (Letter et al., 19831. Reprodluction Diabetic mice of both sexes on all backgrounds will not mate, and females are hypogonadal. Therefore, breeding is accomplished with heterozygotes. To aid in the identification of heterozygous breeders on the C57BL/KsJ and C57BL/6J inbred backgrounds, the db gene has been placed in repulsion with the coat color mutation misty (m). Black, lean mice obtained from a cross of db + / + m heterozygotes are used as breeders, whereas lean mice with grey coats ~ + m/ + m genotype) are discarded. Black mice that become obese at weaning are the presumptive mutants (db + Idb + genotype). When it is desirable to identify presumptive dbidb mice as early as 3 days post- partum, breeding stocks are utlized in which db and m are maintained in coupling (db ml + + J. The mutant pups can be recognized by the absence of pigment in the paws and on the tip of the tail.

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46 IMMUNODEFICIENT RODENTS df (Ames Dwarf) Genetics Ames dwarf (did appeared first in 1961 as an autosomal recessive mutation in a line of extreme nonagouti (ae/ae) mice derived from a cross between Goodale's giant and a pink-eyed stock (Schaible and Gowen, 19611. Although phenotypically similar to the mutation dwarf (dw) (see page 49), df is not allelic with dw. The df mutation, which maps to chromosome 11, is main- tained on an outbred and on the NFR/N inbred backgrounds. Pa thophys lo logy Homozygous Titmice resemble dwldw mice. Growth retardation is observed after 1 week of age, and by 2 months of age they are only one-half the weight of controls. The anterior pituitary lacks cells that produce either prolactin (Barkley et al., 1982) or growth hormone (Duquesnoy and Pedersen, 19811. Ames dwarf mice have a deficit of the T-cell component of the immune system, which becomes apparent by 3 weeks of age. Morphological abnor- malities include depletion of lymphocytes in the thymus-dependent regions of lymph nodes and periarteriolar sheaths of the spleen follicles and a grad- ually progressive lymphopenia. This abnormality culminates in the devel- opment of progressive atrophy of lymphoid tissue, signs of wasting and infection, and early death. Other features of the T-cell deficit are depressed ability of spleen cells to induce graft-versus-host reaction, impaired ability to reject allogeneic skin grafts, and impaired phytohemagglutinin (PHA)- induced blastogenesis. Both dwarf mutations have a similar deficit of T-cell function, although the deficit is more severe in df than in dw mice. There are subtle differences found in the kinetics of the immune response to sheep red blood cells (SRBCs). Although dw mice always develop more feeble plaque-forming cell (PFC) responses compared with control mice, they show an increase in PFC re- sponses with increasing doses of SRBCs. In contrast, df mice do not show a dose-related dependency of immune response to SRBCs. The reason for this difference between the two dwarf mutations is not well understood, and numerous theoretical explanations have been postulated (Duquesnoy and Pedersen, 19811. Husbandry Mice carrying the df mutation should be maintained as described for those carrying dw (see page 491.

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HEREDITARY IMMUNODEFICIENCIES 129 Pathophysiology Homozygous tl rats can be distinguished from their normal littermates by 10 days of age by their smaller size, short snout, and lack of incisors. Periorbital incrustation, possibly caused by defective lacrimal ducts, is com- mon. Bones are thickened and lack marrow cavities. Osteoclasts are rare (Cotton and Gaines, 1974; Marks, 1977), and those present are small and have greatly reduced concentrations of acid hydrolases (Seifert et al., 1988~. Toothless rats are hypophosphatemic and mildly hypocalcemic (Seifert et al., 1988) and respond poorly to exogenous parathyroid hormone (Marks, 19779. Peritoneal macrophages in tlltl rats are decreased 100-fold compared to normal littermates (Wiktor-Jedrzejczak et al., 1981~. In vitro responsiveness of spleen cells to T- and B-cell mitogens exceeds that of normal littermates and appears to be a function primarily of the adherent cell population (Wiktor- Jedrzejczak et al., 19811. Toothless rats are not cured by transplantation of normal bone marrow or spleen cells (Marks, 1977), and skeletal sclerosis cannot be induced by transplantation of mutant spleen cells into normal animals (S. C. Marks, Jr., University of Massachusetts Medical School, unpublished data). Husbandry The it mutation is not lethal; however, a soft diet is essential to compensate for the lack of incisors. The husbandry of this mutant has been reviewed (Marks, 19871. SPF conditions are recommended. Reproduction Homozygotes breed poorly or not at all. The mutation should be maintained by heterozygous matings. C4 Deficiency Genetics A single-component deficiency for the fourth component of complement has been reported in Wistar rats (Arroyave et al., 19741. The mode of inheritance was reported to be autosomal recessive, with heterozygotes ex- pressing 50 percent of normal levels of C4. The gene encoding for the complement fraction C4 has been mapped to the right of RT1.B in the rat MHC (Watters et al., 19871.

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130 IMMUNODEFICIENT RODENTS Pathophysiology Arroyave et al. (1977) found that total hemolytic complement activity (CHso) in these animals was 20 percent of normal. It could be restored to normal by the addition of purified human C4. No C4 inhibitor was found in the sera of affected rats that were capable of responding to the injection of normal rat serum by producing anti-C4 antibodies. Although both female and male rats were deficient, a significantly higher number of males were affected. Husbandry Special husbandry procedures are not required. Reproduction Although no precise mechanism has been identified, the reproductive ef- ficiency of this colony is lower than expected (small litter size). INBRED STRAINS OF RATS BB/Wor anti Other Sublines Genetics The BB Eformerly called BB (BioBreeding) Wistar] rat develops an acute form of spontaneous juvenile insulin-dependent diabetes mellitus (IDDM) resembling human diabetes mellitus type I. The development of this model has been reviewed (Chappel and Chappel, 19831. The gene conferring sus- ceptibility to diabetes has been shown to be associated with the MHC. Spe- cifically, there is a requirement for the RTlU haplotype or a gene in close linkage with the gene coding for this haplotype (Colle et al., 19811. Non- MHC genes are also involved in the susceptibility to diabetes in this strain (Jackson et al., 1 9841. Several features of the syndrome suggest the involvement of the class II u antigens (RTl.B/DU) in the pathogenesis of IDDM (Goldner-Sauve et al., 19861. This is supported by the data of Buse et al. ~ 1984), which are indicative of a restriction fragment length polymorphism (RFLP) in class II MHC genes. On the other hand, Kastern et al. (1984) have demonstrated by RFLP analysis that a BB/Wor-derived subline lacks a major proportion of class I MHC genes. Furthermore, Wonigeit has found that BB/Wor-derived rats express the uv4 haplotype defined by a variation in atypical class I antigens (RT 1. CUV4) (K. Wonigeit, Klinik fur Abdominal- und Transplantationschirurgie der Med

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HEREDITARY IMMUNODEFICIENCIES 131 izinischen Hochschule, Hannover, Federal Republic of Germany, personal communication to H. J. Hedrich, 19884. The presence of the RTl U allele is not in itself sufficient to result in clinical manifestation of the disease. Additional factors are required for overt dia- betes; a susceptibility for the development of insular, periductular, or in- traacinar lymphocytic infiltration in the pancreas has been proposed. This susceptibility is thought to be coded for by the dominant gene Pli (pancreatic lymphocytic infiltration), which segregates independently of RT1 (Colle et al. 19834. Overt IDDM is strongly associated with a genetically controlled depression of circulating T lymphocytes (Guttmann et al., 19831. The nature of the T lymphopenia gene (1) is not yet known, but it has not been linked to the MHC. Whether the expression of the I gene is fortuitous or obligatory for IDDM needs to be verified. Herold et al. (in press) have reported on the results of a cross between an inbred (F25) diabetes-prone BB substrain with an incidence of IDDM of greater than 90 percent and an inbred (F25) diabetes- resistant BB substrain with no diabetes and no lymphopenia. The F1 offspring were normal, but in the F2 generation, the overall incidences of diabetes and lymphopenia were 30 and 27 percent, respectively. Lymphopenia was present in 76 percent of the diabetic rats but in only 5 percent of the nondiabetic animals. Furthermore, the diabetes occurred earlier in nonlymphopenic than in lymphopenic rats. Like et al. (1986b) have demonstrated that diabetes occurs among diabetes-resistant control lines (WA, WB, and WC) without lymphopenia. Several groups are inbreeding BB rats originating from the outbred BioBreeding Wistar colony. Various lines exist with a variable degree of inbreeding and differences in the genetic background, except for the genes determining IDDM (RTl U and Pli, with or without 11. Selective breeding has resulted in a 60-80 percent penetrance in affected substrains. Normoglycemic sublines (diabetes-resistant) with less than 1 percent incidence of IDDM are available. Recently, Guberski et al. (in press) demonstrated that diabetes- resistant BB/Wor rats have a geneks) controlling the age of onset of diabetes. However, the genetic basis of the disease needs further investigation. Pathophysiology The disease is characterized by insulitis (mononuclear cell infiltration of the pancreas), which causes destruction of the islets of Langerhans (Seemayer et al., 19831. Acute clinical signs, including hyperglycemia (greater than 300 mg/dl), nonmeasurable insulin levels, ketoacidosis, hyperglucagonemia, polydipsia, and polyuria, develop between 6 weeks and 6 months of age (average age at manifestation is 3 months). The rats survive only if insulin is administered daily (Chappel and Chappel, 19831.

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132 IMMUNODEFICIENT RODENTS The characteristics of IDDM development in BB rats are suggestive of an autoimmune etiology. The development of overt hyperglycemia can be sup- pressed by immune modulatory techniques, including antilymphocyte serum, cyclosporine A, neonatal thymectomy, or injections of monoclonal antibodies specific to NK cells or CD4 + T lymphocytes (Like et al., 1 979, 1 98 1 , 1 986a; Stiller et al., 19831. Furthermore, it is possible to prevent the manifestation of diabetes mellitus by the application of antibodies directed against class II MHC gene products (Boitard et al., 1985), by bone marrow transplantation (Naji et al., 1983), or by transfusion of whole blood from a nondiabetic subline without prior immunosuppression of the recipient (Rossini et al., 19851. The disease can be adaptively transferred to young, nonaffected, diabetes-prone rats or to immunosuppressed histocompatible rats by using ConA-activated spleen cells from acutely diabetic donors (Koevary et al., 19839. In addition to anti-class II MHC antibodies, it has been shown that multiple autoantibodies react with parietal cells of the gastric mucosa, thyroid colloid, thyroid follicular cells, skeletal muscle, smooth muscle, and nuclear protein (Dyrberg et al., 1982; Elder et al., 1982; Baekkeskov et al., 19841. A Hashimoto-like thyroiditis has also been demonstrated (Sternthal et al., 19811. The presence of autoantibodies directed against lymphocytes is controversial (Dyrberg et al., 1982; Elder et al., 19821. Diabetes-prone rats that develop diabetes mellitus always exhibit a severe impairment of the T-cell system. Both the helper/inducer and the cytotoxic/ suppressor T-cell subsets in peripheral blood and lymphoid tissues are pro- foundly reduced (Bellgrau et al., 1982; Colle et al., 1983; Elder and MacLaren, 19831. An inversion of the ratio of helper/inducer T cells (CD4) to cytotoxic/ suppressor T cells (CD8) occurs in all BB rats by about 60 days of age (Prud'homme et al., 19841. Woda et al. (1986) have shown that diabetes- prone BB rats lack the classic cytotoxic/suppressor T-lymphocyte subset (CD8~. Furthermore, Greiner et al. (1986b) have demonstrated that RT7.1 + T cells are depleted in diabetes-prone rats and that no RT6.1 + T cells are detected in their peripheral tissues. Functional analysis of lymphopenic animals shows a severe deficiency of all T-cell functions analyzed (Jackson et al., 1981; Elder and MacLaren, 1983), depressed skin allograft rejection across MHC and non-MHC barriers (Bellgrau et al., 1982; Kloting et al., 1984), defective proliferative responses to mitogens and to allogeneic cells in MLC (Rossini et al., 1983; Prud'homme et al., 1984), and defective cytotoxic T-cell function as measured by cell- mediated lympholysis (Prud'homme et al., 19881. The number of B cells and serum immunoglobulin levels are no1lllal, and the number of PMNs is increased, apparently in response to various chronic respiratory infections. The in vitro analysis of T-cell functions suggests that most BB sublines

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HEREDITARY IMMUNODEFICIENCIES 133 exhibit immunologically incompetent T cells, which result from a postthymic or peripherally acquired maturational defect (Elder and MacLaren, 19831. It is not clear whether the mechanism of ,Bcell destruction and its pre- vention are the same in lymphopenic and nonlymphopenic (less than 3 percent of diabetic-resistant lines) animals (Like et al., 1986b). Butler et al. (1988) suggest that a deficiency in suppressor cell activity results in an unchecked effecter cell-induced ,Bcell cytotoxicity. NK cells or effecter cells that do not display the classical cytotoxic/suppressor T-cell phenotype might also be involved (Greiner, 19861. Guberski et al. (1988) were able to create an obese animal model of autoimmune diabetes mellitus by crossing Zucker female rats that were het- erozygous for the gene fatty (fal+) to male diabetic BB/Wor (+/+) rats. F1 hybrid females were backcrossed to BB/Wor diabetic males, and diabetic backcross males and females were mated. This latter mating fixed the re- cessive diabetes genes. Progeny from this mating were bred back to F1 hybrids, and progeny were selected that were genotypically diabetic and carriers of the fa gene. These rats served as parents for later inbreeding, which resulted in a new diabetic rat, BBZ/Wor. The BB/Wor diabetic rat resembles the lean BBZ/Wor rat (fal+ or +/+~: both are nonobese; both have insulitis, comparable rates of diabetes, thyroiditis, similar islet cell pathology, and ketosis; and both require insulin therapy. Obese BBZ/Wor rats (falfa) are heavier and have prominent ,~cell hyperplasia and degran- ulation. The obese BBZ/Wor rats are of the RTl U haplotype, are lymphopenic, and have an incidence of diabetes comparable to those of lean BBZ/Wor and BB/Wor rats. The obese BBZ/Wor rats, however, do not require exogenous insulin, most likely because of incomplete destruction of the pancreatic ,l3 cells (Guberski et al., 1 9881. Husbandry Maintaining BB rats is difficult because of their extreme susceptibility to opportunistic infections (Elder and MacLaren, 19831. It is therefore advisable to maintain these animals under laminar-flow, SPF, or gnotobiotic conditions. Gnotobiotic BB rats have the same incidence of diabetes as conventionally housed BB rats. All animals must be weighed several times a week; when weight decreases or fails to increase, the urine should be tested for glucose and ketones using Tes-Tape~ (Eli Lilly & Co., Indianapolis, Ind.) and Ketostix~ (Miles Di- agnostics, Elkhart, Ind.~. When glucosuria is found, plasma glucose should be determined. Once glucose levels are above 250 mg/dl, insulin must be administered daily. The dose is determined by the amount of glucosuria and is usually between 1 and 2.5 units.

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134 IMMUNODEFICIENT RODENTS Reproduction Diabetes in BB/Wor rats is associated with reduced fertility. In males, this appears to be associated with a primary disorder of Leydig's cells, which precedes changes in seminiferous tubules (Murray et al., 1983~. Breeding efficiency in the high-incidence, diabetes-prone lines can be improved by administration of cyclosporine A, which delays the onset of the syndrome (Like et al., 1984), or whole-blood transfusions, which prevent the occur- rence of hyperglycemia (Rossini et al., 19831. Gross anatomical malformations observed in the offspring of diabetic BB rats are exencephaly, dysmaturity of the ossification process in cranial and long bones, and lumbosacral dysgenesis. Pups born to diabetic dams dem- onstrate significant delays in all aspects of motor development during the preweaning period. Control of maternal diabetes (regulation of blood glucose and prevention of ketosis) during pregnancy results in increased litter and fetal size, decreased perinatal mortality, and a significant reduction in the incidence of congenital malformation (Brownscheidle et al., 19831. LOU/C LOU/C rats spontaneously produce an IgG autoantibody that binds and neutralizes rat beta interferon (De Maeyer-Guignard et al., 19841. The titer of this autoantibody increases with age. This strain was selected for its high spontaneous incidence of myeloma, which is first seen at 8 months of age, and it has been postulated that the anti-IFN antibodies contribute to this disease (De Maeyer-Guignard et al., 19841. No studies have been conducted to investigate whether there is an increased susceptibility to infectious agents. GUINEA PIG MUTANTS C2 Deficiency Genetics Deficiency of the second component of complement (C2) in guinea pigs was the first C2 deficiency reported in an animal other than humans (Hammer et al., 19811. The gene for the defect behaves like a rare, silent allele of C2 that is MHC linked and inherited in the same way as normal C2 variants, that is, as an autosomal codominant trait. This mode of inheritance is similar to that in humans (BiKer-Suermann et al., 19811. The C2-deficient allele (C2) is linked to C4S~ and BfF allotypes within the guinea pig MHC.

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HEREDITARYIMMUNODEFICIENCIES 135 Pa thophys to logy C2 deficiency is characterized by a total absence of the second component of complement, as measured by hemolytic C2 and antigenic C2 protein assays. It is the most frequently seen complement deficiency in humans, with an incidence approaching 1 percent (Pettier, 19821. Macrophages in C2-deficient guinea pigs synthesize an abnormal C2-like protein rather than functionally active C2 (Goldberger et al., 19821. C2-deficient guinea pigs show no unique susceptibility to infectious dis- eases. Changes in the vascular permeability of skin induced by injection of the Cls subcomponent of the first component of complement are absent in C2-deficient guinea pigs but are present in C4-deficient guinea pigs, which suggests that the permeability agent is derived from C2 itself (Strang et al., 1986~. The C2-deficient guinea pig is unable to make a good antibody to the T-cell-dependent antigen, bacteriophage 4'X174. The antibody responses are characterized by low titers, failure to detect amplification of the secondary response, and no IgM to IgG switch (Ochs et al., 19861. Humans with C2 deficiency have a high incidence of SLE, discoid lupus, and Henoch-Schon- lein purpura. The C2 deficiency is more common in women than in men, and the incidence of lupuslike syndromes is more common in C2-deficient women than in C2-deficient men. C2-deficient guinea pigs have hypergam- maglobulinemia, anti-DNP antibodies, and rheumatoid factor; however, overt autoimmune disease is not seen (Bottger et al., 1986a). This guinea pig model might be a good model for studying the relationship between C2 deficiency and SLE in humans. Husbandry Special husbandry procedures are not required. Reproduction These animals breed normally. C3 Deficiency Genetics The defect in the third component of complement (C3) arose spontaneously in strain 2 guinea pigs, was expressed in an autosomal codominant fashion, and was not linked to the MHC or to the gene controlling expression of the C3a receptor (Burger et al., 19861.

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136 IMMUNODEFICIENT RODENTS Pa thop hys lo logy Homozygous C3-deficient (C3D) guinea pigs have a functional C3 titer and antigenic activity that is only 6 percent of normal. Serum from these animals has a markedly reduced hemolytic complement and bacteriocidal activity in vitro, and homozygous deficient animals have impaired antibody synthesis to the T-cell-dependent antigen, bacteriophage X174 (Bottger et al., 1986b). In addition, C3D guinea pigs have a defect in isotype switching from IgM to IgG. These in vivo abnormalities in humoral immunity are shared with guinea pigs with deficiencies of C2 or C4 (Bottger et al., 19851. However, in contrast to C2D and C4D guinea pigs, C3D guinea pigs do not have elevated IgM levels, nor do they have circulating immune complexes (Bottger et al., 1986b). Macrophages and hepatocytes from guinea pigs with homozygous C3D synthesize and secrete C3 in normal amounts. When ana- lyzed by sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis and immunoblotting, the product of C3D hepatocytes appeared normal; how- ever, it was greatly reduced in the serum of animals containing the deficiency. The catabolism of normal guinea pig C3 was not elevated in C3D guinea pigs; however, enhanced proteolysis of a defective C3 molecule in C3D animals could not be excluded (Auerbach et al., 19851. Husbandry C3D guinea pigs, unlike humans with a similar deficiency, do not appear to have an increased susceptibility to infectious agents or immune complex disease (Alper and Rosen, 1984; Bottger et al., 1986b). Reproduction No problems have been reported in the breeding of C3D guinea pigs. C4 Deficiency Genetics Deficiency of the fourth component of complement (C4) in guinea pigs is inherited as an autosomal recessive trait with full penetrance (Hyde, 1923; Ellman et al., 1970), resembling the inheritance of C4 deficiency in humans (Ochs et al., 1977; Schaller et al., 19771. Heterozygotes express intermediate levels of C4 (Ellman et al., 19701. It is thought that the deficiency arose from a mutation of a C4-F allele (Shevach et al., 19761.

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HEREDITARY IMMUNODEFICIENCIES 137 Pathophysiology C4 deficiency was first discovered in a strain of guinea pigs whose serum did not lyse antibody-sensitized horse erythrocytes (Moore, 19191. The defect was discovered independently in a colony of outbred Hartley guinea pigs at the National Institutes of Health (Ellman et al., 19701. Guinea pigs with a C4 deficiency are unable to activate complement by the classical pathway (Hammer et al., 1981), because C4 is critical for the development of both C3 and CS convertase (Muller-Eberhard, 1975J. This model has been used extensively to demonstrate the amino acid sequence of pro-C4 (the precursor to C41. The structure, function, and quan- titation of guinea pig C4 have recently been reviewed (Quimby and Dil- lingham, 19881. The discovery of C4 deficiency in guinea pigs provided a good model in which to study the activities of the classical and alternative pathways of complement activation (Frank et al., 19711. The genetic control and biosynthesis of C4 have also been studied in the C4-deficient guinea pig (Colten and Frank, 1972; Colten, 19831. Recent experiments with cell-free biosynthetic systems have shown that guinea pig C4 is synthesized as the single-stranded precursor pro-C4 (200,000 dalton), which, prior to its release from the polysome, is converted to the three-chain C4 molecule (Hall and Colten, 19771. Hall and Colten (1977) failed to detect the presence of pro-C4 in hepatocytes of C4-deficient guinea pigs, and Colten (1983) has hypothesized, based on hybridoma studies, that the deficiency is due to a posttranscriptional defect in the processing of C4 precursor RNA to mature C4 messenger RNA. The C4-deficient guinea pig has also been used to evaluate the role of the classical pathway during various infections (Diamond et al., 1974; Gelfand et al., 19784. Early studies showed that animals with suspected C4 deficiency were more susceptible to Salmonella (formerly Bacillus) choleraesuis infec- tion than were normal animals (Moore, 19191. C4-deficient guinea pigs are more susceptible to the lethal effects of endotoxin injection (May et al., 19721. Resistance to the ixodid tick Dermacentor andersoni (Wikel, 1979) and to Candida albicans (Gelfand et al., 1978) and Cryptococcus neoformans (Diamond et al., 1974) infections is normal in C4-deficient animals. It can be inferred, therefore, that there is an intact alternative pathway of comple . . . . .. . ment act~vat~on ~n guinea pigs with t nese ~ntect~ons. Ellman et al. (1971) have demonstrated a slight but significant defect in the ability of C4-deficient guinea pigs to make an antibody response to DNP- bovine y-globulin. The antibody response to the T-cell-dependent antigen bacteriophage 4'X174 is clearly abnormal in C4-deficient guinea pigs. Not only is the IgM response depressed, but there is also no evidence for im- munologic memory (Ochs et al., 1978, 19861. C4-deficient guinea pigs have signs of polyclonally stimulated antibody synthesis, circulating rheumatoid

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138 IMMUNODEFICIENT RODENTS factors, and anti-DNP antibodies, which suggests immune complex disease (Bottger et al., 1986a). Despite the latter observation, there have not been any particular problems associated with infectious agents in conventionally reared colonies (Peltier, 19821. Husbandry Special husbandry procedures are not required. Reproduction These animals breed normally (Peltier, 19821. HAMSTER MUTANTS nu (Nude) Genetics Nude (nu) is a recessive mutation that arose spontaneously in the breeding colony of outbred Syrian hamsters at the Institut de Recherches Scientifiques sur le Cancer, Villejuif, France (Haddada et al.- 19821. Genetic studies involving these animals have not been conducted. Pa thophys to logy Nude hamsters, like nude mice and nude rats, are hairless and have only a rudimentary thymus. At 1 month of age, decreased numbers of mature lymphocytes are found in lymph nodes and the spleen. Spleen cells do not proliferate in response to the mitogens ConA, PHA, LPS, and protein A, and the level of serum IgG is reduced. In contrast to nude mice and nude rats, nude hamsters have no higher NK cell activity than do normal hamsters. Spontaneous tumors do not arise frequently; however, a study has shown that 1 of 11 animals developed an immunoblastic sarcoma in the mesenteric lymph node at 8 months of age (Loridon-Rosa et al., 19881. Injection of simian virus 40-transformed B-lymphoma cells induces tumor development in nude hamsters but not in immunocompetent controls (Loridon-Rosa et al., 1988). Husbandry No definitive studies have been performed to evaluate the susceptibility of nude hamsters to various infectious agents; however, animals housed under conventional conditions live 10-12 months and often develop skin disorders

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HEREDITARY IMMUNODEFICIENCIES 139 after 8 months. Because the immunologic defect in nude hamsters appears to be similar to that of nude mice and rats, SPF conditions are recommended for their maintenance. Reproduction Female homozygous nude hamsters are fertile but are unable to feed their young; therefore, nude hamsters are maintained by mating homozygous nude males with heterozygous female siblings. C6 Deficiency Genetics The genetic control of the deficiency of the sixth component of complement (C6) in hamsters is incompletely understood. Pathophysiology C6 deficiency in hamsters was described by Yang and coworkers (1974~. Serum from affected hamsters was incapable of reconstituting C6-deficient rabbit or human sera but did reconstitute sera depleted of Clr, C2, and C4. The C6 deficiency was confirmed by titration of isolated complement com- ponents from normal and deficient hamster sera. C6-deficient hamster sera did not have altered immune adherence or phagocytic functions. Hamsters with the deficiency had a high incidence of proliferative enteritis (transmis- sible ileal hyperplasia). Husbandry Special husbandry procedures are not required. Reproduction These animals breed normally.