Primary immune response in grafted cells. Dissociation between the proliferation of activity and the proliferation of cells.

The primary immune response elicited by host antigens in a grafted population of immunologically competent cells has been compared in conditions where the same dose of parental cells were grafted simultaneously to F(1) hybrid embryos of 13 or 17 days of age. The enlarged chimeric spleens harvested 4 days later were analyzed for donor cell proliferation by using the sex chromosomes as karyological markers, and for proliferation of immunological activity by means of transfer to secondary hosts of the same genotype. Whereas the total number of dividing donor cells were on the average 16 times higher in 17-day than in 13-day hosts, the recovery of immunological reactivity showed a 6- to 7-fold difference in the opposite direction. The experiments cast doubt on the proposition that cellular proliferation is necessary for development of a primary immune response. They suggest that there exists an alternative way in which a primary immune response may unfold from involving a few to involving a much larger number of cells.

The frequency of antigen-sensitive cells in tissue transplantation. A commentary on clonal selection.

Graft-vs.-host (GVH) reactions were performed in chicken embryos by intravenous injection of adult chicken blood dilutions, and the result was scored by weighing the spleens as well as by karyological identification of host and donor metaphases. From the frequency of detectable signs of GVH reaction when low doses of donor cells were injected into hosts containing one foreign allele of the B locus it is concluded that 1-2% must be a minimum estimate for the frequency of antigen-sensitive cells of a given specificity in this system. It is not found possible to reconcile the findings with the strictest form of clonal selection which postulates a single receptor specificity per clone of antigen-sensitive cells.

The dynamics of red cell chimaerism in histoincompatible parabiosed mice.

A new technique for the quantitation of isoenzymes was applied to assess the proportions of red cells in the circulation of parabiosed mice. Two parent-F1 hybrid combinations showing phosphoglucose isomerase polymorphism were examined at successive stages of parabiosis and after separation. Once red cell populations became mixed, 3 days after union, the ratio of red cell phenotypes was never significantly different from one partner to the other, although parental red cells became predominant after about 20 days. However, F1 hybrid red cells could always be detected. After separation of parabiosed mice there was a return to the original composition although this took longer than would be expected on the basis of reported red cell life span. Packed cell volume measurements indicated that a parental polycythaemia and F1 hybrid anaemia developed in one strain combination but not in the other. Evidence was adduced to support the hypothesis of a difference in red cell flux being responsible for the generation of this polycythaemia-anaemia.

Tissue repopulation during cure of osteopetrotic (mi/mi) mice using normal and defective (We/Wv) bone marrow.

Resorption of petrotic bone in osteopetrotic (mi/mi) mice was brought about by transplantation of bone marrow to X-irradiated recipients. In an attempt to learn more about the donor cell line involved in this process, both normal and defective marrow were used. The consequent repopulation of the lympho-myeloid complex was monitored by isoenzymes of glucose phosphate isomerase. The progress of normal marrow grafts was contrasted with that of a defective marrow (We/Wv). Despite the observation with We/Wv marrow showed reduced ability to form colonies in the spleen of an irradiated recipient, this marrow was as effective as normal marrow in inducing resorption of petrotic bone. The primordial stem cell for the osteoclast (haematopoietic stem cell?) is thus not a CFUS. Chimaeras with resolution of osteopetrosis by We/Wv bone marrow may exhibit erythropoiesis from residual stem cells of the host but leucocytes and platelets from the donor.

Resolution and relapse of osteopetrosis in mice transplanted with myeloid tissue of variable histocompatibility.

Osteopetrotic microphthalmic mice (mi/mi) were treated by injections of suspensions of myeloid tissue, newborns i.p., and weanlings i.v. Donated syngeneic material effected permanent cure of oteopetrosis provided that the dose was large enough (10(8) cells of bone marrow). H-2-compatible allogeneic bone marrow was initially as effective, but relapse ensued in immunocompetent mice. H-2-incompatible marrow was ineffecitve except in one set of newborn tolerant mice. Total body X-radiation in sublethal doses to recipients allowed permanent cure with H-2-compatible, and, in one circumstance, with H-2-incompatible marrow in smaller doses. The best results were obtained after lethal irradiation and the smaller dose of marrow. Results were checked by chromosome assay demonstrating that cure or relapse was correlated with permanent take or rejection, respectively, of a transplant in a recipient's bone marrow. Retention of donor lymphocytes alone was not associated with effective bony resorption; the candidate cell line for effectiveness remains the haematopoietic stem cell-monocyte-tissue phagocyte.

The origin of osteoclasts.

We are satisfied from studies with mi mi osteopetrotic mutant mice that osteoclasts arise from the myeloid tissue of bone marrow and not as formerly proposed from osteoprogenitor cells. Grafts of compatible normal myeloid cells cure the osteopetrosis by the substitution of the qualitatively defective osteoclasts with normal ones. Nevertheless it is still not fully clear through what cellular cascade this is effected. Current opinion would favour the pathway from pluripotent haematopoietic stem cells to circulating monocytes to tissue macrophages with ultimate fusion to form multinucleate osteoclasts. However, it is recorded that osteoclasts differ from macrophage polykaryons of inflammatory tissue not only in certain subcellular characteristics but in absence of Fc and C3 receptors. We can explain this as due to development through a specialised line of osteoclast precursors independent of conventional macrophages, if current unpublished experimental studies confirm the transfer to osteoclasts of the additional "beige" marker incorporated into grafted material.

Bone absorption and the immune system.

The absorption of bone in Grüneberg microphthalmic mice and the op op osteopetrotic rat can be stimulated by an injection of compatible bone marrow, and complete resorption can occur. It is probable that the bone-absorbing osteoclast responsible is derived from a haematopoietic stem cell resident in the bone marrow (BM). Maintenance of the resorption depends on the survival of donor cells which has an important implication for clinical therapy in man. No evidence was found that the thymus played a leading role in the mechanism of bone absorption in the Grüneberg microphthalmic mouse, or in the op op rat.

Resorption of bone.

The cell-system responsible for resorption of bone is now considered to be a derivative of haematopoietic bone-marrow, not skeletal connective tissue. Consideration of mutant mice and rats, with defects of bone resorption giving osteopetrosis, suggests that the primary defect is of the professional scavengers, the mononuclear-phagocyte system, failing to recognise effete bone. To explain associated defects of thymic lymphocytes it is postulated that the mononuclear-phagocyte system may be activated to a major or minor extent by professional recognisers, thymic lymphocytes, as happens in some inflammatory reactions.

The fate of allogeneic grafts of intact bone marrow in immunologically tolerant recipients and after abrogation of the tolerance.

In ossicles derived from grafts of compatible intact bone marrow in the subcapsular renal site the ectopic bone remains of donor provenance but the haemopoietic elements are partially replaced with tissue by host cells derived from stem cells migrating from the blood. Ossicles derived from incompatible intact bone marrow grafted in immunologically tolerant recipients are morphologically identical to those derived from compatible intact bone marrow. On abolition of the tolerance the allogeneic ectopic bone and donor microenvironment for haemopoiesis became manifest: haemopoietic marrow, even the host elements, was rapidly lost, bone disappearing rapidly if recently established, slowly if long established. Provided the state of immunological tolerance persists, the histoincompatible bone and microenvironment of the donor co-exist with marrow derived from the circulating haemopoietic stem cells of the tolerant recipient.

Abrogation of BCG-contact induced tumour inhibition by silica: implications for the mechanism of action.

The outgrowth of tumours from inocula of syngeneic rat tumour cells injected in admixture with BCG (Glaxo strain) is suppressed, the tumour cells apparently being destroyed in the milieu of the granulomatous reaction to the mycobacteria. The possible candidacy of macrophages as the major "effector cell" component was examined in experiments conducted in vivo using silica, a selective macrophage toxin. Intraperitoneal (i.p.) administration of this agent abolished contact-induced inhibition of BCG against three transplanted rat sarcomas, although for two of them the abrogatory effect could not be achieved without prior in vitro cultivation of the tumours. It is suggested that i.p. silica not only destroys macrophages within the cavity but accomplishes systemic depletion to the extent that granulomata contain insufficient macrophages for tumour rejection. This deficit may be compensated to a variable extent by the presence of host macrophages within the initial bacterial: tumour cell inoculum, but in the absence of these cells, total abrogation of the inhibitory effect of BCG is the invariable outcome in silica-treated hosts.

Donor origin of the in vitro hematopoietic microenvironment after marrow transplantation in mice.

Bone marrow stroma from radiochimeric mice was established in culture. The polymorphic enzyme glucose phosphate isomerase (GPI) was used to determine the proportions of donor and recipient present in the original bone marrow and in cultured stroma. Bone marrow initially containing 95% donor GPI, when cultured and subsequently passaged for up to 8 weeks remained about 70% donor GPI. We conclude that many cultured stromal cells are donor derived in our radiochimeras and these are probably of hematopoietic origin.

Failure of thymic grafts to stimulate resorption of bone in the Fatty/Orl-op rat.

An infusion of compatible normal bone marrow stimulated bone resorption in both unthymectomized and thymectomized Fatty/Orl-op osteopetrotic rats. Bone resorption was not stimulated by compatible normal thymic grafts in either unthymectomized or thymectomized mutants. It is concluded that the thymus is not fundamental in the cure of osteopetrosis in this strain of osteopetrotic rat and that the defect lies in myeloid rather than thymic regulation of osteoclastic function.

A study of osteoclasts on calvaria of normal and osteopetrotic (mi/mi) mice by vital staining with acridine orange.

A novel staining procedure for enumerating osteoclasts on neonatal mouse calvaria with the vital fluorescent dye acridine orange is described. It has the advantage over Barnicot's neutral-red method in that the nuclei and cytoplasm of the osteoclast are stained differentially. The osteopetrotic calvarium (mi/mi) has fewer multinucleate osteoclasts than its normal counterpart (mi/+) and they are differently distributed. The osteopetrotic calvarium has more mononucleate cells which stain like osteoclasts with acridine orange than the normal calvarium and these cells also are differently distributed. These mononuclear cells may be mononuclear osteoclasts or their precursors. These observations suggest that the defect resulting in this osteopetrosis lies with osteoclast differentiation.

Osteopetrosis in the Grüneberg (mi) mouse can be cured by cultured allogeneic bone marrow.

H2 compatible allogeneic bone marrow cultured for four weeks was effective in inducing bone resorption when injected intravenously into osteopetrotic mi mouse. The active cell line or its progenitors can therefore be established in culture and is probably derived from a haematopoietic stem cell (HSC).

Evidence for osteoclast production in mixed bone cell culture.

Osteoclastlike cells (OLCs) were recognized by their morphological, histochemical, and behavioral characteristics. These characteristics were initially observed in freshly isolated osteoclasts when neonatal rat bone cells were introduced into culture. Epithelioid cells proliferated during the first week to form a confluent layer. The cells' alkaline phosphatase content and ability to produce elevated levels of cyclic adenosine monophosphate in response to parathyroid hormone (PTH) led us to tentatively describe them as osteoblastlike cells. When a second addition of neonatal bone cells was made onto this confluent layer, OLCs were produced, their numbers increasing to a maximum 7 days later and dying out 3 weeks later. Mononuclear cells with similar characteristics to these multinuclear OLCs were observed and their production and disappearance closely paralleled that of the OLCs. Fusion of OLCs was observed. The conditions under which OLCs were produced are described.