R. E. Billingham

The melanocytes of mammals.

Melanocytes may be regarded as unicellular, pigment-secreting glands, largely though not entirely confined to the epidermis, of which they are a constant though often un-recognized cellular component. Their numerical incidence, and their branching form, with numerous dichotomizing processes, are such as to form a reticular system within the epidermis. Evidence is evaluated that they are a race or lineage of cells sui generis and form a self-maintaining system within the epidermis. Their product-melanin granules-is secreted directly into the cytoplasm of neighboring Malpighian cells upon which the processes of the melanocytes terminate in the form of small swellings or end-caps. The several types of branched cells demonstrable within the epidermis by various techniques are discussed in relation to their identity as living melanocytes, or as effete cells of this lineage which, having lost or discharged their pigment, participate in the general out-ward movement of epidermal cells to be lost from the skin surface. The color variants of skin and hair, including spotting and albinism, are the direct result of melanocyte activity, and have been shown to be determined by the action and interaction of multiple genes, some of which operate via the milieu in which the pigment cells reside and others of which appear to act intracellularly and to control the biochemical steps of melanogenesis. The problem of pigment spread is discussed and related to the anatomy of the melanocyte system and to the co-existence of variant types of melanocytes within a single individual.

Studies on the reaction of injected homologous lymphoid tissue cells against the host.

With some combinations of inbred strains of mice, such as CBA -+ A and vice versa, it has been found that the intravenous injection of newborn animals with a dosage of 4 to 10 million living homologous adult spleen cells will induce a high degree of tolerance of subsequent skin homografts from the donor strain in a great proportion of the subjects.lS Inoculation of the newborn animals by the intraperitoneal route is less effective in conferring tolerance, and the subcutaneous route is completely ineffective, probably because cells introduced by this route do not reach the developing immunologically Lompetent tissues of the host soon enough. At birth the mouse, unlike the raL,3 is almost a t the end of the tolerance-responsive phase of its life. With this method of inducing tolerance in mice, a completely unexpected complication was encountered in certain strain combinations; for example, C57 + A and AU + A, in which all injected animals died within 2 or 3 weeks after an initial period of normal growth and apparent well-being. The first indication of disease was cessation of growth, usually accompanied by diarrhea; death followed within a few days. Uninjected litter mates, it may be emphasized, remained perfectly healthy and unaffected by the disease that was killling their injected brothers and sisters. With other strain combinations, although most of the mice appeared to be suffering from this disease, its severity was variable: a few of the victims died soon after injection following an “acute” attack; others suffered from what appeared to be a chronic, sublethal attack that retarded their growth so severely that it was by no means unusual to find animals with body weights of no more than 5 to 7 gm. at 50 to 70 days of age (see FIGURE 1). Fortunately, from the point of view of obtaining tolerant animals, a variable proportion of the mice, depending upon the particular strain combination, either failed to show any clinical symptoms of this disease or recovered from what appeared to be a very mild attack. As m B L h 1 shows, with nearly all strain combinations tested a fairly high proportion of the surviving mice were found to be tolerant when challenged with a skin homograft from the donor strain of the neonatal spleen cell inoculum. Unfortunately, because of their small size and feeble condition, only a small proportion of the runts could be test grafted, but all those that survived this operation proved to be fully tolerant for as long as they lived. The subject matter of this paper is an investigation of the etiology of this peculiar disease, referred to descriptively as “runt disease,” which is associated with the inoculation of newborn mice with adult homologous spleen cells. Some of the work described in this paper was carried out in collaboration with Leslie Brent of University College, London, England.

Quantitative studies on the induction of tolerance of homologous tissues and on runt disease in the rat.

The principle that rats may be rendered tolerant of homologous tissue grafts following preor early postnatal exposure to living homologous cells is now well established (Woodruff and Simpson, 1955; Billingham et al., 1956; Lengerovh, 1957; Egdahl et al., 1958). It is also known that “runt” disease (Billingham, 1958; Billingham and Brent, 1959) not infrequently occurs in this animal, as in the mouse, if the inoculum employed to induce tolerance contains a high proportion of immunologically competent cells (Woodruff and Sparrow, 1957; Egdahl et al., 1958; Billingham and Silvers, 1959~). Their size renders rats much more convenient subjects than mice for detailed analyses of quantitative aspects of the induction of tolerance and the pathogenesis of runt disease: for example, large amounts of bone marrow can be obtained from a single animal, thoracic ducts of rats can be cannulated to obtain almost pure lymphocytes in considerable quantities, and daily samples of blood may be taken from very young animals for exact hematological analyses without prejudicing their well-being. Unfortunately, however, these advantages hitherto have been more than counterbalanced by the lack of the necessary isogenic strains, so that present knowledge derives largely from work conducted on mice. The recent availability of 2 highly inbred strains of rat, the Lewis (albino phenotype) and the B.N. (chocolate), differing so widely with respect to their origins that the degree of genetic disparity between them is likely to be very great (Billingham and Silvers, 19593), has terminated this unsatisfactory state of affairs and thus facilitated the investigations that form the subject matter of this paper. Destruction of skin homografts exchanged between adult members of the B.N. and Lewis strains is invariably complete within 13 days, and the median survival time (MST) of Lewis

The role of regional lymphatics in the skin homograft response.

The importance of an intact lymphatic circulation for the evocation of skin homograft sensitivity was investigated using guinea pigs of two inbred strains. Circular flaps of skin were cut from the flank with the preservation of a slender vascular bundle to maintain viability. The wounds were closed and the flaps were housed in plastic dishes fixed to the underlying skin. Dye injections showed that this procedure effectively deprived these quasi-isolated skin flaps of lymphatic communications with their hosts. Skin homografts placed in beds prepared in the flaps did not undergo rejection, during the period of flap viability, (19–57 days), although control homografts in intact skin were invariably rejected in 8–10 days. Skin homografts made into flaps borne by presensitized animals were promptly destroyed, demonstrating that the absence of lymphatic drainage does not prevent the expression of extant homograft sensitivity. It is believed that the interruption of lymphatic drainage from these artificial privileged sites breaks the afferent are of the immunologic reflex with regard to skin homografts.

THE NEOGENESIS OF SKIN IN THE ANTLERS OF DEER

To most biologists it is almost if not quite axiomatic that adult mammals cannot regenerate normal skin in areas where its entire thickness has been lost or destroyed. The very existence of a specialized branch of surgery that deals largely with the making good of cutaneous lesions is testimony of the incompetence of man to generate new skin. The natural healing of a wound involving the loss of the full thickness of the skin is the result of two more or less concomitant processes. First, the loss of the substance of the corium is made good by the formation of highly vascular granulation tissue that is progressively resurfaced by epithelium of migratory origin from the wound margins. Second, as a result of tensile forces generated within the wound area itself, and almost certainly within the substance of the granulation tissue or its derivatives, the normal skin from the original wound margins becomes drawn inward, and there is a consequent reduction in the extent of the lesion (Billingham and Medawar, 1955; Abercrombie et al., 1956; Billingham and Russell, 1956a; Watts et al., 1958). In regions of the body where the skin is loosely knit to the underlying structures and is therefore freely mobile, as in the trunk of nearly all mammals, contracture usually brings about complete closure of the wound by apposition of its original edges, so that only a narrow scar remains. The loss of skin substance that this process entails is gradually made good by a compensatory expansion of the surrounding skin by a process of intussusceptive growth-that is, the formation of new tissue upon or within the framework provided by pre-existing tissue (Billingham and Medawar, 1955 ; James, 1959). However, in areas of the body where the skin is firmly attached to the underlying tissues (as in the skin over most of the body of man and in the skin covering the ear cartilage of most animals) contracture cannot proceed to completion. There is a permanent residual scar of variable extent, comprising tough, relatively inelastic, white fibrous tissue covered by epithelium. The formation of this stable scar cannot be regarded as a regeneration since nothing has been regenerated: scar tissue is certainly not true skin that has been formed anew. I t differs profoundly from normal skin with respect to the configuration of its dermoepidermal interface and its fibrous architecture. There is a deficiency of elastic fibers and a tendency for its collagen fibers to be disposed in a horizontal plane rather than in the usual three-dimensional packing. This is responsible for its poorly developed resilience. Hairs are not normally regenerated anew, although it is commonly said that regeneration of sweat glands may take place in scar tissue in man. * Some of the work reported in this paper was supported by Grant C-3577 from the National Cancer Institute, Public Health Service, Bethesda, Md. The Wistar Instilute, Philadelphia, Pa.

Influence of the Ag-B locus on reactivity to skin homografts and tolerance responsiveness in rats.

SUMMARY The case with which rats of one strain can be rendered tolerant of skin grafts from another strain is correlated with their Ag-B genotypes and not with the median survival time of skin grafts exchanged between them. Thus, although adult Lewis recipients reject Ag-B-compatible Fischer skin grafts as promptly as they reject Ag-B-incompatible BN or DA skin grafts, Lewis recipients are much more easily rendered unresponsive of Fischer skin grafts than of BN or DA grafts. Where Ag-B compatibility prevails, tolerance is inducible with relatively low dosages (≤1 million) of cells originating from all components of the lymphohematopoietic tissue system, including the thymus. On the other hand, when donor and host differ at this locus not only are much higher numbers of cells required to induce tolerance, but there are wide disparities in the tolerance-conferring capacity of different cell types. Bone marrow is superior to spleen which, in turn, is better than lymph nodes; thymocytes are practically ineffective. Evidence is also presented which suggests that the facility with which tolerance can be abolished by transfer of isologous cells is a function of the dosage of the inoculum used to induce tolerance. The importance of the Ag-B locus in determining susceptibility to runt disease is also considered.

The h-y transplantation antigen, a y-linked or sex-influenced factor.

IN mice and rats there is one exception to the rule that grafts are always accepted between members of an isogenic population. This applies when females are challenged with skin and other tissues from male donors1–6. The ability of females to reject these grafts varies from strain to strain. For example, while virtually 100 per cent of C57BL/6 (hereafter C57) females and about 75 per cent of A strain females reject male skin isografts, CBA and AU strain females usually accept such grafts. This interstrain diversity occurs despite the fact that the specificity of the Y antigen or “Y factor” is apparently the same in male mice of all stocks7. The basis for this variability stems from two factors: the first is the genotype of the female which determines her capacity to react against male skin and the second is the genetic background of the male which influences the expression of the Y antigen8. Thus the complete penetrance of the Y factor in C57 mice is due to the females of this strain being relatively strong reactors against male skin isografts, and is not because the antigen is strongest in this strain. Indeed, that the antigen is weaker in C57 males than in CBA males follows from the fact that (CBA × C57)F1 hybrid females reject grafts from CBA males more frequently than from C57 males8.

Transplantation Immunity of Gestational Origin in Infant Rats

Comparison of the survival times of homografts of BN skin on 3-day-old Lewis rats born of mothers of the same isogenic strain with those of BN grafts on infant Lewis hosts that had developed in an F1 (Lewis x BN) hybrid, gave no evidence of maternally induced tolerance as a result of development in an antigenically alien environment. On the contrary, the significantly shorter median survival time of the grafts on the hybrid-derived Lewis group suggests that sensitization had occurred as a consequence of natural exposure during gestation to small numbers of maternal cells.

Hamster immune responses in infectious and oncologic diseases

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STUDIES OF TRANSPLANATION IMMUNITY IN HAMSTERS

Unlike all other common laboratory mammals, Syrian hamsters (Mesocricetus aurafus) appear to be unique in that they will often accept homografts of malignant tissues and, on occasion, even heterografts of both normal and malignant tissues (Toolan, 1955; Foley and Handler, 1957; Pierre, Verney, and Dixon, 1957). A detailed study has therefore been undertaken to determine whether the Syrian hamster’s response to skin homografts also differs from that of other animals. The adult Syrian hamsters employed were obtained from four different closed colonies in Britain; each of these was known to have been completely isolated for an extended period, although none of them had been deliberately inbred by successive brother-to-sister matings (TABLE 1). Male animals were used for most of this work. In all of the experiments to be described, two full-thickness skin grafts about 7 or 8 mm. in diameter, from which all adherent fascia1 tissue had been removed, were transplanted to beds of the appropriate size prepared in the skin of the chest of the recipient. Primary inspection of the grafts was carried out on the eighth postoperative day, and subsequent inspections \wrc made at 2or 3-day intervals for the first 3 weeks and less frequently thereafter. Animals whose grafts survived were kept under observation for a t least 100 days. The survival times of the homografts were assessed on the basis of their outward appearance (Billingham and Medawar, 1951), and confirmed as required by the histological examination of biopsy specimens. The majority of the homografts exchanged between hamsters derived from the same colony were accepted by their hosts and regenerated perfectly normal hair crops just as if they were autografts. These grafts remained in perfect condition until the experiments were terminated after 100 days or longer. The numbers of animals that fully accepted their intracolony homografts are given in FIGURE 1. However, the grafts on somc of the animals did become inflamed and edematous and were infiltrated by lymphocytes; their complete destruction followed within a few days of the onset of these typical symptoms of incompatibility. The breakdown of the grafts on all except one of the animals that rejected its homografts was complete within 3 weeks. In the exceptional animal of the CB colony whose grafts survived for about 35 days, and in other recipients in later experiments to be described below, the onset oC the reaction was delayed and was of the mild, chronic type. There was a prolonged weak inflammation of The j a t e oJ inlracolony homografts.