Calcium homeostasis: how bone solubility relates to all aspects of bone physiology.

This perspective challenges the bone research community to study a new concept of calcium homeostasis and determine how it affects all aspects of bone physiology and disease. The concept started with Neuman's discovery that the apparent supersaturation of calcium in the extracellular fluid (ECF) could be explained by the presence of non-collagenous proteins on the surfaces of bone. His discovery opens the door to a new field of bone research and raises the question of how his result affects other aspects of bone physiology and pathology? The purpose of this perspective is to challenge the bone field to determine the significance of these findings. The report lists a few areas that need inquiry and supplies premises that need to be tested.

Calcium homeostasis: solving the solubility problem.

This report summarizes the evidence that the control of the concentration of free calcium ions in body fluids is centered at mineralized bone surfaces. This process involves an increase in the solubility of bone mineral produced by the non-collagenous proteins existing in the bone extracellular fluid (ECF) and on the adjacent surfaces of bone. The result is a basic equilibrium level produced in the absence of parathyroid hormone (PTH), which is well above the solubility of bone mineral. The effect of PTH is to increase the solubility of bone mineral still further, but the mechanism by which the hormone acts is unknown. The lining cells of the bone contain receptors for PTH and can be observed to respond to this hormone, but the relationship between this response and the increased solubility of bone remains to be discovered. Further research in this field is strongly urged.

Evidence that tolerance to cultured thyroid allografts is an active immunological process. Protection of third-party grafts bearing new antigens when associated with tolerogenic antigens.

We studied the tolerance phenomenon that develops in long-term recipients of cultured thyroid allografts. Allogeneic mouse thyroids were cultured under hyperbaric oxygen or acidic conditions and then transplanted beneath the kidney capsule of C57BL/6 recipients. Donors differed from the recipients in minor antigens alone, major histocompatibility complex antigens alone, or both. At 35-77 weeks after the first cultured graft, recipients received two more cultured grafts under the capsule of the opposite kidney and were immunized with donor spleen cells (SC). At 5 weeks after the second transplantation, we observed that whereas second grafts carrying new antigens alone were rejected, second grafts carrying new antigens in association with antigens in the first graft were significantly protected. In another set of experiments, normal mice became tolerant to cultured allografts after 2 weeks in parabiosis with tolerant individuals. Tolerant mice showed reduced specific in vivo and in vitro cytotoxic T lymphocyte responses. However, the frequency of CTL precursors of tolerant mice was the same as in normal mice. The reduced in vitro CTL responses were restored to normal levels by the addition of a lymphokine rich medium. Also, we observed that the injection of specifically activated immune SC caused the rejection of cultured allografts in normal but not in tolerant recipients. We conclude that the tolerance that develops in recipients of cultured allografts is an active immunological process that affects the activation and effector function of CTL.

Major histocompatibility complex antigen expression of parenchymal cells of thyroid allografts is not by itself sufficient to induce rejection.

H-2d thyroids cultured in oxygen at normal atmospheric pressure (suboptimal culture) were grafted into H-2b mice. Some of these tissues were cultured with recombinant mouse gamma-interferon (rIFN), and they expressed high levels of major histocompatibility complex antigens before grafting. Three weeks later, no difference in the rate of rejection of MHC-induced grafts was observed as compared with uninduced tissues (50% of each group). Fresh (uncultured) grafts were uniformly rejected in less than 2 weeks. Also, H-2d thyroids, freed of donor leukocytes by preculture in hyperbaric oxygen and more than 1 year parking in normal H-2b recipients, were incubated with and without rIFN, and then grafted into normal H-2b mice; 100% acceptance was observed in both groups regardless of the expression of allo-MHC molecules on thyroid cells. In another set of experiments, using grafts with a single antigenic difference at the MHC locus (bm1 into B6), graft rejection was observed only when the recipients were immunized with donor spleen cells and fresh tissues were implanted. In the same immune recipients, cultured and MHC-induced thyroids grafted in the opposite kidney were, in general, not rejected. These results demonstrated that the expression of allo-MHC molecules on graft cells was not by itself sufficient to engender tissue immunogenicity. This supports our previous hypothesis that the main effect of tissue culture is the inactivation of passenger leukocytes. MHC antigens appear to be immunogenic only when properly presented by these cells.

Role of lymphokine in islet allograft rejection.

Primed CD8 T cells transfer allograft immunity to an established islet allograft. However, the process is inhibited by cyclosporine, suggesting that lymphokine production is required for islet graft rejection. The alloreactive T cell clone L3 will transfer allograft immunity, and this process is also sensitive to CsA. The L3 clone produces gamma-interferon and tumor necrosis factor but not IL-2 and IL-3. It follows therefore that the latter lymphokines are not required for the rejection process. Pretreatment of islet tissue with gamma-IFN prior to grafting increases the density of the class I major histocompatibility complex antigen on the islet tissue, and CsA can no longer block the destruction of this MHC-induced tissue by primed alloreactive T cells. We conclude that gamma-IFN, and possibly TNF, act cooperatively with cytotoxic function in the process of islet allograft rejection.

T cells can be cytotoxic without making interleukin 2: a model of separate pathways of induction.

The thoracic duct lymphocytes from rats previously injected with ultraviolet-light-irradiated allogeneic lymphocytes were grown for 4 days with alloantigen, with or without Con A-induced lymphokine factors, and then for 3 days with the lymphokines alone. They were then tested for their cytoxicity and for their capacity to make interleukin 2 (IL-2) in response to antigen. The results show that T helper cells specific for both class I and class II antigens of the major histocompatibility complex were removed from the circulation by the injection of ultraviolet-irradiated alloantigen. However, precursors of cytotoxic cells remained and appeared to lose their OX-19 markers during activation. We have interpreted the results by using a speculative model that involves separate pathways of induction to cytotoxicity and IL-2 synthesis. We propose that the OX-19 marker is associated with the interleukin 1 receptor and that the latter is required for the IL-2 production pathway but not for activation to cytotoxicity.

Synergism between minor and major histocompatibility antigens in the rejection of cultured allografts.

Thyroid lobes cultured in hyperbaric oxygen were transplanted into recipients that differed from the donor in major histocompatibility complex (MHC) antigens alone (2 strain combinations), minor histocompatibility antigens alone (5 strain combinations), or both (5 strain combinations). Significant foci of lymphocytic infiltration, referred to here as a delayed type hypersensitivity reaction, were seen only if there were minor antigenic differences, and a significant number of rejections occurred only if there were both minor plus MHC antigenic differences. Thus, these results demonstrated a synergism between some minor and MHC histocompatibility antigens in the rejection of cultured allografts. Possible mechanisms of this synergism are discussed.

Macrophage activation: dissociation of cytotoxic activity from Ia-A antigen expression.

Peritoneal macrophages were obtained from DBA/2 mice that were untreated or after the injection of bacillus Calmette-Guerin (BCG), thioglycollate broth, proteose-peptone broth, or gamma-irradiated P-815 tumor cells. These macrophages were "activated" to become cytotoxic for a fibroblast cell line (L 929) by the addition of lymphokines (LKs), lipopolysaccharide (LPS), or fibroblast interferon (IFN-beta), and the expression of I region-associated antigens (Ia-Ad) on the macrophages was examined both before and after activation. Thioglycollate-elicited macrophages became Ia-A+ when activated by LKs, but they remained Ia-A- when activated by LPS or IFN-beta. Resident macrophages and proteose-peptone-elicited macrophages remained Ia-A- when activated with LKs. Macrophages from BCG-infected mice were both Ia-A+ and cytotoxic for tumor cells without further treatment. In contrast, macrophages from mice injected with gamma-irradiated P-815 mastocytoma cells were Ia-A+ but not cytotoxic, and these macrophages could not be made cytotoxic by incubation with LKs. The cultured macrophage-like cell lines P388D1 and WEHI-3 became Ia-A+ after incubation with LKs, and this treatment amplified the cytotoxicity of both cell lines. We conclude that a number of factors are important in determining whether Ia-A expression accompanies macrophage activation and that Ia-A is irrelevant as a surface marker for macrophage activation.

The failure of a major histocompatibility antigen to stimulate a thyroid allograft reaction after culture in oxygen.

The lymphocytic infiltration found in multiple cultured BALB/c thyroids placed in the same C57BL/6 recipient was found to be highly correlated and the high variability between animals was not influenced by the number of lobes. It was concluded that the variable infiltration was largely due to host factors. Because a similar correlation was found between BALB/c and C3H grafts, the response to a minor antigen common to these two strains was suggested as a cause of the infiltration. When the response of C57BL/6 mice to cultured B6.C (H-2d) and BALB.B (H-2b) grafts was compared, a synergism between major and minor antigens was suggested. However, when the time of culture was increased from 48 to 60 h and the response of BALB/c and C57BL/6 mice were compared with identically cultured B6.C (H-2d) grafts, a striking difference between major and minor antigens was observed. None of 10 such grafts in C57BL/6 recipients (major antigens only) showed any infiltration, whereas 8 out of the 9 grafts in BALB/c recipients (minor antigens only) were infiltrated.

Rejection of stable cultured allografts by active or passive (adoptive) immunization.

Injection of live lymphoid cells of donor strain or immune cells of recipient strain resulted in rejection of previously stable cultured mouse thyroid allografts. The results are interpreted to indicate that a cultured graft is relatively ineffective in activating recipient lymphocytes but is capable of maintaining them in an activated state and serving as a target for them once they are activated.

Dimethyl sulfoxide prevents DNA nicking mediated by ionizing radiation or iron/hydrogen peroxide-generated hydroxyl radical.

Eighty percent of the single-strand DNA breaks induced by gamma-irradiation were prevented by the hydroxyl radical (.OH) scavenger dimethyl sulfoxide (Me2SO); CH4 was generated in the process as a product of the interaction of .OH and Me2SO. In contrast, Me2SO completely blocked DNA nicking by an iron/H2O2 system which produces .OH but smaller amounts of CH4 from Me2SO. Because Me2SO prevented DNA breaks from the more efficient iron/H2O2 system but only blocked 80% of irradiation-mediated nicking, the results suggest that .OH is responsible for 80% of the DNA single-strand breaks and the remaining 20% is due to interactions not involving .OH.