Conditioned medium of fetal mouse long bone rudiments stimulates the formation of osteoclast precursor-like cells from mouse bone marrow.

We studied the influence of skeletal tissue on expression of the osteoclastic phenotype in the mouse, in vivo and in vitro. In various soft and hard tissues of adult and fetal mice the distribution of mono- and multinucleate cells showing tartrate-resistant acid phosphatase was studied, using enzyme histochemistry on undecalcified plastic sections. Cells with strong TRAcP activity were only observed in mineralized tissues. Multinucleate TRAcP cells were exclusively found in close correlation with mineral resorption. In fetal bones mononuclear TRAcP cells appeared in the surrounding soft tissue prior to osteoclast formation. In addition, conditioned media of fetal long bone rudiments (FBCM) increased the number of mononuclear cells showing strong TRAcP activity in 7 d cultures of adult bone marrow. FBCM stimulated DNA synthesis in TRAcP cells and induced their multi-nuclearity. Pretreatment with FBCM increased the capacity of bone marrow cultures to form osteoclasts in coculture with fetal bone rudiments. However, FBCM did not change the number of cells with tartrate-sensitive acid phosphate activity (TSAcP cells). The activity of FBCM was heat labile and was not detectable in CM of killed bones. CM of embryonic mouse fibroblasts which contains M-CSF activity, strongly increased the number of TSAcP cells but reduced the number of TRAcP cells. These data suggest that fetal mouse bone tissue induces the differentiation of osteoclast precursors. In addition, fetal bone rudiments but not embryonic fibroblasts seem to produce a factor(s) which stimulate(s) the formation of cells showing characteristics of osteoclast precursor cells. Osteoclasts and macrophages seem to have different growth requirements, indicating that they represent separate cell lines which may nevertheless derive from a common progenitor.

Role of mineralizing cartilage in osteoclast and osteoblast recruitment.

Periost-free, live and/or devitalized cartilaginous long bone rudiments of fetal mice were transplanted under the renal capsule of adult syngeneic mice to study the role of cells and intercellular matrix in the recruitment and formation of osteoclasts and osteoblasts, both identified by means of enzyme- and immunohistochemical methods. Live bone rudiments recruited host-derived osteoclasts within 5 days after transplantation. Osteoblasts developed as rapidly as osteoclasts and participated in the modeling of the rudiments into hemopoietic bone marrow containing ossicles. Devitalized bone rudiments, killed before osteoclastic invasion had occurred, did not recruit osteoclasts or osteoblasts, and were not resorbed up till 35 days after transplantation. Co-transplantation of live and devitalized bone rudiments however resulted in osteoclastic resorption of the killed rudiments, starting 9 days after transplantation. Again the live rudiments were modeled into ossicles. Devitalized bone rudiments which had been invaded by osteoclasts before killing and transplantation, did recruit host osteoclasts, but at a slower rate than live rudiments, and depending on the number of resorption sites at the time of transplantation. Osteoblasts were not formed. These data suggest that in developing long bones chondrocyte activity is involved in the recruitment of osteoclasts as well as osteoblasts. Matrix components diffusing from resorbing surfaces seem to be involved in osteoclast recruitment.

Growth of osteoclast precursor-like cells from whole mouse bone marrow: inhibitory effect of CSF-1.

We studied the growth of mononuclear phagocytes (MPs) and mononuclear osteoclast precursor-resembling (OCP-like) cells from freshly isolated whole mouse bone marrow. Expression of tartrate-resistant acid phosphatase (TRAcP) served as a general marker for the identification of OCP-like cells. Bone resorbing capacity of the cultured cells was studied in a coculture assay with periost-free fetal bone rudiments. Freshly isolated mouse bone marrow contained approximately 30 OCP-like cells and 1100 MPs per 10(6) nucleated bone marrow cells. OCP-like cell numbers did not increase in suspension cultures containing macrophage-colony stimulating factor (CSF-1), in contrast to the number of MPs which increased strongly. OCP-like cell numbers however did increase in monolayer cultures, which also allowed anchorage-dependent growth of bone marrow fibroblasts. Strongest increase of OCP-like cells occurred in monolayer cultures in the absence of CSF-1. Dermal fibroblasts of fetal mice did not enhance OCP-like cell growth. OCP-like cell density was strongly correlated with the number of osteoclast nuclei formed in cocultures with periost-free bone rudiments. These data indicate that mononuclear cells, cytochemically and functionally resembling osteoclast precursors, may be grown from mouse bone marrow. CSF-1 inhibited growth of OCP-like cells, indicating that osteoclast precursors differ from mononuclear phagocytes in growth requirements.

Characteristics of osteoclast precursor-like cells grown from mouse bone marrow.

Cells resembling mononuclear osteoclast precursors (OCP-like) were grown from mouse bone marrow and subsequently studied for expression of macrophage-specific surface antigens (F4/80 and Mac-1), phagocytosis activity, and DNA synthesis. Expression of tartrate-resistant acid phosphatase (TRAcP) activity served as general marker for OCP-like cells. Mononuclear phagocytes, characterized by expression of tartrate-sensitive acid phosphatase (TSAcP) activity, were also grown from mouse bone marrow and were compared with OCP-like cells. TRAcP-positive OCP-like cells showed low values for F4/80 and Mac-1 expression, as well as low phagocytosis activity. Macrophages (TSAcP+) showed very high values for expression of both surface antigens and for expression of phagocytosis activity, while osteoclasts (TRAcP+), reported to lack Ag F4/80, were also negative for Mac-1. DNA synthesis in OCP-like cells was low and resembled the values found in macrophages. Chase experiments after nuclear pulse labeling indicated that TRAcP-positive OCP-like cells developed from immature proliferating progenitor cells which did not yet show TRAcP activity. These results show that TRAcP-positive OCP-like cells have characteristics of both mononuclear phagocytes and osteoclasts. We propose that OCP-like cells are relatively late stages in an osteoclast-specific cell lineage which diverges from the mononuclear phagocyte lineage.

Development and use of shell-less quail chorio-allantoic-membrane cultures to study developing skeletal tissues; a qualitative study.

A technique is described for in vitro culture of the quail embryo from the 1st to the 18th day of development. The embryos are cultured in Teflon hammocks, suspended in glass supports and kept in a humidified atmosphere at 36.5 degrees C. The quail CAM is used as support and cell source for developing non-quail cartilage and bone. The quail cells can be identified histologically and easily recognized by Feulgen-staining which is demonstrated in the presence of quail chondro- or osteoclasts in a mouse long bone rudiment cultured on the CAM.

Demonstration of tartrate-resistant acid phosphatase in un-decalcified, glycolmethacrylate-embedded mouse bone: a possible marker for (pre)osteoclast identification.

Fixed, undecalcified mouse long bones were embedded in glycol methacrylate (GMA), sectioned, and incubated for acid phosphatase in the presence or absence of tartrate, to investigate the feasibility of tartrate-resistant acid phosphatase as a histochemical marker for osteoclast identification. Naphthol AS-BI phosphate was used as the substrate and hexazonium pararosanaline as coupler. Cytocentrifuge preparations of mouse, rat, and quail bone marrow or frozen and GMA sections of mouse splenic tissue were used as controls to specify acid phosphatase activity. After adequate fixation, acid phosphatase activity sensitive to tartrate inhibition (TS-AP) was demonstrated in macrophages from spleen, bone marrow, and loose connective tissue surrounding bone rudiments. Acid phosphatase activity resistant to tartrate inhibition (TR-AP), was detected in multi-nuclear osteoclasts and in some mononuclear cells from bone marrow and periosteum. In cytocentrifuge preparations and frozen sections of mouse spleen, TR-AP was demonstrated after simultaneous incubation with substrate and tartrate. In GMA sections, however, TR-AP could only be demonstrated after pre-incubation with tartrate before application of substrate. We suggest that histochemical demonstration of TR-AP versus TS-AP on GMA-embedded bone sections by means of a pre-incubation method can be used as an identification marker of (pre)osteoclasts. Plastic embedding is recommended for its excellent preservation of morphology and enzyme activity.

The thymus in "bare lymphocyte" syndrome: significance of expression of major histocompatibility complex antigens on thymic epithelial cells in intrathymic T-cell maturation.

Thymic biopsies from two patients with combined immunodeficiency and defective expression of HLA class I and class II antigens on blood mononuclear cells ("bare lymphocyte" syndrome) were investigated. This made possible an evaluation of the significance of HLA antigen expression in a detailed (immuno)histologic study. Both thymuses showed a normal lobular architecture with distinct cortex-medulla areas, well-differentiated epithelium, including ultrastructurally defined subtypes, and Hassall's corpuscles. Normal numbers of lymphoid cells were present and normal T-cell phenotype was found. Using anti-HLA-A,B,C antisera, confluent staining of the medulla (stroma and lymphocytes) was observed. One of the thymuses was found to be negative for HLA class II antigen expression: the other revealed only HLA-DR positivity of nonlymphoid cells in the medulla. These cells were not of epithelial nature as judged from double staining with anti-keratin antibody. There was no expression of HLA-DC/DS. These observations differ from findings in the normal thymus, wherein epithelial cells in the cortex carry HLA class I and class II antigens, and epithelial cells in the medulla express HLA class I, and for a minor part class II antigens. The results indicate a normal sequential acquisition of T-cell differentiation antigens in the thymus of both cases. It is suggested that the expression of HLA class I and class II antigens on epithelial cells in the normal thymus cortex does not play a significant role in the sequential acquisition of differentiation antigens on T lymphocytes.

Heterogeneity and age dependency of human thymus reticulo-epithelium in production of thymosin components.

The specific compartmentalization of intrathymic lymphoid cells in different steps of T-cell maturation and the involvement of thymosin components in these steps prompted us to evaluate the localization of epithelial cells synthesizing various thymosin components. Thymosin fractions 5 and 6 were found in epithelial cells throughout the thymus cortex and medulla. Thymosin alpha 1 and beta 4 were observed in subcapsular and perivascular epithelial cells, and thymosin alpha 1 was observed in the medullary epithelium. Thymosin alpha 7 was exclusively found in Hassall's corpuscles and adjacent epithelial cells. We conclude that the organ localization of epithelium synthesizing different thymosin components is associated with the organ localization of intrathymic T-cell maturation steps. The involution of the human thymus, starting during the third decade of life, has been related to the age-dependency of thymus humoral factors in the circulation. However, quantitative data on humoral factor synthesizing epithelial cells are lacking. We found a strong intensity of thymosin fraction 6 expression by epithelium in thymus tissue from donors in the early postnatal period (2-4 months of age) and from all four donors aged 20-30 years. This phenomenon was paralleled by a high number of thymosin fraction 6 positive cells per unit area.

Heterogeneity of epithelial cells in the human thymus. An ultrastructural study.

To evaluate interrelationships among epithelial cells, and between morphology and function in the microenvironment, we studied the ultrastructural morphology of epithelial cells in sections of human thymus from donors aged 2 months to 31 years. Six types of epithelial cells were observed: "subcapsular-perivascular" (type 1); "pale" (type 2); "intermediate" (type 3); "dark" (type 4); "undifferentiated" (type 5); and "large-medullary" (type 6). Cells of types 2, 3 and 4 were found throughout the organ. The type-2 to -4 epithelial cells may represent various stages in a differentiation process. In this, type-2 cells are very active and type-4 cells are possibly degenerating elements. Type-4 cells can also contribute to Hassall's corpuscles. Type-5 cells were located mainly in the cortico-medullary region and showed the morphological characteristics of undifferentiated elements. Type-6 cells were located exclusively in the medulla and displayed characteristics of cellular activity. Small Hassall's corpuscles consisted of type-6 epithelial cells; in larger corpuscles many nuclei of type-6 cells were found. Cells of types 2 and 6 contained tubular structures (diameter approximately 20 nm). Concerning the function of thymus epithelial cells, the features associated with protein synthesis observed in cellular types 2 and 6 make them likely candidates for humoral factor-producing and/or secreting elements. In addition, type-2 and -3 cells in the cortex appear to contribute to a special pattern of epithelium-lymphocyte interaction ("thymic nurse cells"), as demonstrated by the intracytoplasmic location of lymphocytes in the epithelial cells. The various steps in intrathymic T-cell maturation occur at locations in a microenvironment composed of morphologically distinct epithelial cells.

Identification and in situ localization of the "thymic nurse cell" in man.

The observation of the "thymic nurse cell" (TNC), a reticuloepithelial cell with intracytoplasmic lymphocytes, in suspension of murine thymic tissue prompted us to investigate the existence of this cell in cell suspension, as well as in tissue sections of the human thymus. TNC-like cells were enriched in suspension by enzymatic disintegration of thymic tissue and 1 X G sedimentation over 50% fetal calf serum gradients. TNC-like cells were negative for lysosomal enzymes: in this respect, as well as in light microscopic morphology, the cells were different from tissue macrophages with intracytoplasmic lymphocytes. In electron microscopy, TNC-like cells showed reticuloepithelial characteristics. In 1-micron tissue sections, clusters of lymphocytes with a possible reticuloepithelial nucleus were observed close to blood capillaries in the cortical area. Ultrastructural analysis confirmed the epithelial nature of this cell, as well as its location adjacent to blood capillaries. We concluded that there is in situ existence of TNC in man. This observation enables studies on the role of TNC in intrathymic T cell maturation.