Defective support of S1/S1d splenic stroma for humoral regulation of stem cell proliferation

Humoral regulation of CFUs proliferation was investigated in S1/S1d mice characterized by a stromal defect, which severely limits in situ proliferation of in vivo colony-forming cells (CFUs). Injection of LPS-W evoked a large enhancement of CFUs numbers in the spleen of normal +/+ mice. S1/S1d mice were found to be refractory to low doses of LPS-W (up to 15 micrograms/mouse) and to have a diminished response to high doses (up to 150 micrograms). Serum transfer experiments showed that S1/S1d mice are not defective in the early elaboration (6 h) of a humoral factor (SHSF), which mediates the LPS-induced splenic stem-cell accumulation. In a serum-free in vitro system post-LPS S1/S1d and +/+ sera induced a similar degree of CFUs proliferation, indicating the ability of S1/S1d mice to produce normal levels of stem-cell-activating factor (SAF). Transfer of potent post-LPS serum from normal mice evoked a poorer splenic CFUs accumulation in S1/S1d mice as compared to normal +/+ littermates. The population size of splenic stem cells in S1/S1d mice parabiosed with normal +/+ mice also showed a limited increase in response to LPS-W injection. This diminished in vivo response of S1/S1d mice was not due to a decreased sensitivity of their CFUs for SAF, since S1/S1d and +/+ CFUs showed similar survival rates in vitro in the presence of SAF. We propose that the defective response of S1/S1d mice to LPS-induced humoral regulators is due to a nonmigratory component of the S1/S1d splenic stroma, which limits splenic CFUs proliferation either by a short-range inhibitory activity or by a deficiency of a local stimulatory activity or nutrient unlike SAF or SHSF, which might act in synergy with SAF.     

Bone marrow origin of mast cell precursors in mesenteric lymph nodes of mice

Concentration of mast-cell precursors in the mesenteric lymph node of (WB X C57BL/6)F1 hybrid mice (WBB6F1) were evaluated by a limiting dilution method. Cells from WBB6F1-+/+ mice were injected directly into the skin of WBB6F1-W/Wv mice which genetically lack tissue mast cells. Concentrations of mast-cell precursors were calculated from the proportion of injection sites at which mast cells appeared. Although immunization with horse serum significantly increased the concentration of mast-cell precursors in the mesenteric lymph node, the concentration in the lymph node remained about 10% that observed in the peripheral blood mononuclear cells. Since the bone marrow origin of mast-cell precursors in the mesenteric lymph node was demonstrated by using giant granules of beige (C57BL/6-bgj/bgj) mice as a marker, the immunization seemed to increase the migration of bone-marrow-derived mast-cell precursors from the peripheral blood to lymph node.     

Mast-cell precursors in various haematopoietic colonies of mice produced in vivo and in vitro

S ummary . We have examined mast-cell precursors in various in vivo and in vitro haematopoietic colonies of mice. Cells from haematopoietic colonies of (WB x C57BL/6)F₁- +/+ mouse origin were injected into the skin of the congeneic W/W^v mice which are genetically depleted of tissue mast cells. The appearance of mast-cell clusters at the injection site indicated the presence of mast-cell precursors in the injected cell suspension. More than 40% of 12 d exogenous spleen colonies and 14 d in vitro mixed colonies contained mast-cell precursors, but only a small proportion of 7 d exogenous spleen colonies, 9 d in vitro erythroid bursts or 14 d large in vitro neutrophil-macrophage colonies contained mast-cell precursors. Neither transient endogenous erythroid spleen colonies examined at the fifth day nor 7 d in vitro neutrophil-macrophage colonies contained mast-cell precursors. Among 12 d spleen colonies mast-cell precursors were more frequent in the predominantly neutrophil than the predominantly erythroid colonies but 14 d in vitro mixed colonies, in which erythroid cells were predominant, contained mast-cell precursors more frequently than 14 d in vitro neutrophil-macrophage colonies. Therefore, differentiation of mast cells seems to be independent of either the neutrophil-macrophage or erythroid cell lineages.     

The granulopoietic effect of human urinary colony stimulating factor on normal and cyclophosphamide treated mice

A practically endotoxin-free colony stimulating factor from human urine (CSFHU) was prepared and its granulopoietic effect on normal and cyclophosphamide treated mice was examined. When normal C57BL/6N mice were injected intraperitoneally with 2.5 X 10(6) units/kg of the CSFHU daily for a 5-day period, the numbers of progenitor cells (CFUC) in the femur and spleen were significantly increased. The CFUC in the femur and spleen reached a maximum at day 3 (270%) and day 5 (250%) after the initial injection, respectively. The increase in number of CFUC in the femur exhibited a dose-dependency with respect to the CSFHU and a significant increase was observed even at 4 X 10(5) U/kg (P less than 0.05). However, neither granulocytosis nor monocytosis occurred in normal C57BL/6N mice injected with the CSFHU. In cyclophosphamide induced leukopenic C3H/HeN mice, daily injections of the CSFHU at 2.5 X 10(6) U/kg for 5 days stimulated the restorative granulocyte production (P less than 0.05) as well as the CFUC recovery in both the femur and spleen. These findings suggested that the CSFHU might be involved in granulocyte production in vivo.     

Macrocytic megakaryocytes in cultures of S1/S1d bone marrow

The thrombocytopoietic system of S1/S1d mice is characterized by macromegakaryocytosis and megakaryocytopenia, but the mechanisms responsible for reciprocal abnormalities of megakaryocyte number and size are unknown. These mice have a genetically determined abnormality of their hemopoietic microenvironment that can, in part, be reproduced as abnormal adherent stromal cells in bone marrow cultures. Cultures of bone marrow were therefore done to determine if the megakaryocytic abnormalities of S1/S1d marrow would also be reproduced in them. Cultures composed entirely of S1/S1d cells showed persistent macrocytosis of megakaryocytes when compared with cultures of normal +/+ marrow. At various times of culture, there were also reduced numbers of megakaryocytes, total cells, and granulocytes in the supernatants of S1/S1d cultures. Mixed cultures of S1/S1d and +/+ cells yielded inconclusive findings. The fact that macromegakaryocytosis occurred in cultures of S1/S1d marrow strongly suggests that its in vivo determinants were, in part, reproduced in culture.     

Development of large numbers of mast cells at sites of idiopathic chronic dermatitis in genetically mast cell-deficient WBB6F1-W/Wv mice

The normal skin and other tissues of adult mast cell-deficient WBB6F1- W/Wv or WCB6F1-Sl/Sld mice contain less than 1.0% the number of mast cells present in the corresponding tissues of the congenic normal (+/+) mice. As a result, genetically mast cell-deficient WBB6F1-W/Wv or WCB6F1-Sl/Sld mice are widely used for studies of mast cell differentiation and function. We found that mast cells developed at sites of idiopathic chronic dermatitis in WBB6F1-W/Wv mice and that the number of mast cells present in the skin of WBB6F1-W/Wv mice was proportional to the severity of the dermatitis (in ear skin, there were 33 +/- 4 mast cells/mm2 of dermis at sites of severe dermatitis v 9 +/- 3 at sites of mild dermatitis, 0.8 +/- 0.3 in skin without dermatitis, and 100 +/- 7 in the normal skin of congenic WBB6F1-+/+ mice; in back skin, the corresponding values were 2.0 +/- 0.6, 1.1 +/- 0.9, 0.025 +/- 0.025, and 26.2 +/- 3.2). The development of mast cells was a local, not systemic, consequence of the dermatitis. Thus, WBB6F1-W/Wv mice with severe dermatitis lacked mast cells in skin not showing signs of dermatitis and also in the peritoneal cavity, stomach, cecum, and tongue. Idiopathic chronic dermatitis was not associated with the local development of mast cells in WCB6F1-Sl/Sld mice, a mutant whose mast cell deficiency is due to a mechanism distinct from that of WBB6F1-W/Wv mice. These findings may have implications for understanding the nature of the mast cell deficiency in WBB6F1-W/Wv and WCB6F1-Sl/Sld mice and for the use of these mutants to analyze mast cell differentiation and function.     

Suppressive effect of Sl/Sld mouse embryo-derived fibroblast cell lines on diffusible factor-dependent proliferation of mast cells

Two modes of mast cell growth are present, one dependent on diffusible growth factors (interleukins [IL] 3 and 4) and another dependent on contact with fibroblasts. The 3T3 fibroblast cell lines derived from WCB6F1-+/+ mouse embryos supported the proliferation of cultured mast cells (CMC), whereas the 3T3 fibroblast cell lines from WCB6F1-Sl/Sld mouse embryos did not. To investigate the relationship between growth factor-dependent and fibroblast-dependent growths of mast cells, we cocultured CMC and 3T3 fibroblasts in the presence of diffusible growth factors. WCB6F1-+/+ mouse embryo-derived 3T3 cells did not affect the growth factor-dependent proliferation of CMC, but WCB6F1-Sl/Sld mouse embryo-derived 3T3 cells significantly suppressed the proliferation. Close cell-to-cell contact was necessary for the suppression. The NWS1 fibroblast cell line was established from the spleen cells of an adult WBB6F1-+/+ mouse. Although the NWS1 cell line had no supporting effect on the proliferation of CMC in the absence of diffusible growth factors, it did not suppress the proliferation of CMC induced by the growth factors. The present result suggests that a product of mutant Sl genes may be involved in the suppressive activity of WCB6F1-Sl/Sld mouse embryo-derived 3T3 cells.     

Hybrid resistance to parental bone marrow-derived cultured mast cells in the skin but not in the peritoneal cavity of (WB X C57BL/6)F1-W/Wv mice

When cultured mast cells of (WB X C57BL/6)F1-+/+(WBB6F1-+/+) and WB-+/+(WB) mice were directly injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, mast cell clusters appeared at the injection sites. Although in vitro colony-forming ability is comparable between cultured mast cells of WB mice and those of WBB6F1-+/+ mice, the number of WB mast cells necessary for the appearance of mast cell clusters in the skin of WBB6F1-W/Wv mice was significantly larger than the number of WBB6F1-+/+ mast cells. In spite of the presence of such an apparent hybrid resistance in the skin of WBB6F1-W/Wv mice to mast cells of the WB parent, both WB and WBB6F1-+/+ mast cells grow in the peritoneal cavity of WBB6F1-W/Wv mice with comparable efficiency. This is a demonstration of the tissue-related (nonrecirculating) expression of hybrid resistance against nonmalignant hematopoietic cells.     

Phorbol 12-myristate 13-acetate-induced development of functionally active mast cells in W/Wv but not Sl/Sld genetically mast cell-deficient mice

The normal skin and other tissues of adult genetically mast cell-deficient WBB6F1-W/Wv or WCB6F1-Sl/Sld mice contain less than 1.0% the number of mast cells present in the corresponding tissues of the congenic normal (+/+) mice. We previously reported that mature dermal mast cells developed locally in the skin of W/Wv, but not Sl/Sld, mice at sites of chronic idiopathic dermatitis. We now report that the repeated application of phorbol 12-myristate 13-acetate (PMA) to the ear skin of either W/Wv or +/+ mice induces both dermatitis and a striking and dose-dependent increase in the number of dermal mast cells. The number of dermal mast cells at sites treated for 6 weeks with 5 micrograms PMA, three times per week, was 39 +/- 7/mm2 and 305 +/- 34/mm2 for W/Wv and +/+ mice, respectively; the corresponding values for vehicle-treated skin were 1.5 +/- 1.0/mm2 and 145 +/- 8/mm2, respectively. The PMA-induced dermal mast cells in W/Wv mice appeared mature by morphology, stained with the heparin-binding fluorescent dye, berberine sulfate, and were competent to express IgE-dependent passive cutaneous anaphylaxis responses. The development of mast cells was a local, not systemic, effect of PMA treatment. PMA treatment also induced dermatitis in both WCB6F1-Sl/Sld and +/+ mice, but was associated with increased numbers of dermal mast cells only in the WCB6F1(-)+/+ mice. PMA treatment had no detectable effect on the ability of bone marrow-derived cultured mast cells to survive in the skin of Sl/Sld mice. These findings establish a convenient model system for analyzing factors associated with the development of endogenous populations of mast cells in genetically mast cell-deficient W/Wv mice.     

Comparison of platelet production in two strains of mice with different modal megakaryocyte DNA ploidies after exposure to hypoxia.

Thrombocytopenia develops with prolonged exposure to hypoxia. Although decreases in megakaryocyte numbers due to hypoxia have been well documented, the effects of hypoxia on megakaryocyte DNA content have not been reported. In this study, megakaryocytopoiesis and platelet production were compared in both C3H mice (whose megakaryocyte modal ploidy class is 32N) and C57/BL mice (whose modal ploidy class is 16N), by enclosure in cages covered with silicone-rubber membranes. After equilibration, O2 levels inside the cages were 6%-7%. Hematocrits, platelet counts, platelet sizes, percent 35S incorporation into platelets, megakaryocyte size and number, and megakaryocyte DNA content of mice were measured before and at various days after hypoxia. Although hematocritis increased and platelet counts decreased in both strains of mice with time in hypoxic chambers, megakaryocyte and platelet responses of C3H mice differed from those of C57/BL mice in several respects; hematocrits of C3H mice were higher and platelet counts were lower than those in C57/BL mice. C3H mice produced larger platelets than C57/BL mice in response to hypoxia. Total circulating platelet counts (TCPC) and total circulating platelet masses (TCPM) of both mouse strains showed similar biphasic responses, that is, elevated TCPC and TCPM on days 2-4 and decreased values after 6-14 days of hypoxia. However, hypoxic C3H mice had lower TCPC on days 4-14 and lower TCPM on days 10-14 of hypoxia than C57/BL mice. Both C3H and C57/BL mice had decreased megakaryocyte numbers at 6-10 days of hypoxia, but only C3H mice had decreased numbers of megakaryocytes at day 14. Elevated megakaryocyte size was observed in both mouse strains at day 14 of hypoxia. However, after hypoxia, C3H mice showed a greater depression in megakaryocyte number and a larger increase in megakaryocyte sizes than did C57/BL mice. C3H mice maintained 32N as the modal megakaryocyte DNA content through day 10 of hypoxia, but 64N was the modal megakaryocyte DNA content at day 14; 16N remained the modal megakaryocyte DNA content in hypoxic C57/BL mice. Hypoxic C3H mice had an increase in 16N megakaryocytes after 6 days of hypoxia, followed by an increase in the proportion of 64N cells at 14 days compared to values of untreated C3H control mice. Hypoxic C57/BL mice had an increased proportion of 16N cells at 6 days but a decreased proportion of 32N cells at 14 days. These studies demonstrate that the decreased platelet production resulting from prolonged exposure to hypoxia is primarily the result of decreased differentiation of hematopoietic precursors into the megakaryocyte lineage rather than decreased megakaryocyte DNA content, because higher ploidy classes actually increase as thrombocytopenia becomes more severe. Stem cell competition could explain the findings of reduced platelet production and increased red blood cell production in both strains of mice after exposure to hypoxia.     

Development of hematopoietic spleen colonies in nonirradiated genetically normal mice.

The question as to whether prior irradiation or injection of cytotoxic drugs is essential for the development of spleen colonies was examined in genetically normal mice. Mixtures of lymph node and bone marrow cells from C57BL mice were injected into (C57BL X CBA-T6T6) F1 hybrid mice without pretreatment. Hematopoietic nodules were observed in the spleens of F1 hybrid mice killed 18 days after injection. The average number of nodules increased linearly with increased numbers of injected bone marrow cells. Hematopoietic stem cells (CFU-S) and dividing cells in the nodules were shown to be of C57BL origin. Histologic examination showed that erythroid cell colonies predominated over granulocytic cell colonies. These results suggest that any kind of treatment that causes the depletion of CFU-S in the spleen of hosts would provide a suitable environment for the production of colonies by transplanted CFU-S.     

Haematological Effects in Mice of Partially Purified Colony Stimulating Factor (CSF) Prepared from Human Urine

S ummary . Single injections of partially purified human urine preparations containing 10 000–20 000 units of colony stimulating factor (CSF) produced a monocytosis and polymorph leucocytosis in neonatal C57BL mice which was maximal at 48 hr. Adult C57BL mice developed a monocytosis, and although no polymorph leucocytosis developed, labelling studies suggested an increased rate of granulopoiesis. In adult mice the injected CSF preparations did not alter total bone-marrow cellularity but increased the percentage of primitive granulocytes and caused an increase in the number of in vitro colony-forming cells and granulocytic cluster-forming cells. The preparations also increased the number of bone-marrow cells able to initiate colony and cluster formation immediately after culture of the bone-marrow cells in agar. The results suggest that CSF in vivo may stimulate both granulopoiesis and monocyte formation in an action similar to its action in agar cultures of bone-marrow cells.     

The preleukemic state of mice reconstituted with Mixl1-transduced marrow cells

Murine granulocytic cells, in becoming leukemic, need to acquire enhanced self-generation and a capacity for autocrine growth stimulation. Mice transplanted with bone marrow cells transduced with the Mixl1 homeobox gene develop a very high frequency of myeloid leukemia derived from the transduced cells. Preleukemic mice contained a high frequency of transduced clonogenic granulocytic cells. They exhibited an abnormally high capacity for self-replication and could generate immortalized granulocytic cell lines that remained absolutely dependent on either GM-CSF or IL-3 and were not leukemic. Organs from mice repopulated by marrow cells transduced either with Mixl1 or the control murine stem cell virus vector exhibited a capacity to produce IL-3 in vitro, activity being highest with the lungs, marrow, bladder, and thymus. Supporting evidence for the in vivo production of IL-3 was the frequent development of mast cells in the marrow. Overexpression of Mixl1 appears capable of inducing an abnormal self-renewal capacity in granulocytic precursors. Aberrant production of IL-3 was not present in the continuous Mixl cell lines and was therefore not in itself likely to be a leukemogenic change but it could support the enhanced survival and proliferation of the Mixl1 granulocytic populations until a final leukemogenic mutation occurs in them.     

Tolerance to the granulocyte-releasing and colony-stimulating factor elevating effects of endotoxin

Injection of Salmonella typhosa endotoxin into either CF1 or C57bl/6J mice leads to prompt increases in serum colony-stimulating factor (CSF). Repeated injections of endotoxin result in a dose-related hyporesponsiveness or tolerance to this effect. Tolerance is seen after either intravenous (i.v.) or intraperitoneal (i.p.) routes of administration or challenge and occurs after one to two preinjections. Cross-tolerance to heterologous endotoxin (Escherichia coli) was also shown. This cross-tolerance is complete immediately after cessation of preinjections, but partial at later time intervals. Levels of a serum inhibitor of colony growth were decreased in tolerant mice, although this decrease is not statistically significant. Tolerant mice injected with endotoxin release granulocytes from the bone marrow normally, in spite of the absence of a CSF response. This suggests that neutrophil releasing activity (NRA) and CSF are separate entities. A marked marrow granulocytic hyperplasia develops after 7 or 20 days of endotoxin injections, despite the tolerance to the CDF-elevating effect of endotoxin. This granulocytic hyperplasia could still be mediated by serum CSF increases. A negative medullary feed-back secondary to the repetitive release of marrow granulocytes, however, is an equally plausible mechanism for the stimulation of granulocyte production. It is also possible that the decrease in serum inhibitors played a role in the sustained increase in granulopoiesis seen here.     

Inhibition by cholera toxin of clonal growth of murine granulocyte/macrophage progenitor cells in soft agar cultures

Proliferation in vitro of the committed granulocyte-macrophage progenitor cell (CFUC) is inhibited by cholera toxin (CT) in a dose-dependent manner. This inhibition is reduced and counteracted by higher doses of colony stimulating factor (CSF), an obligatory growth stimulator of CFUC. Mixing of CT with its specific receptor, the monosialoganglioside, GM1, before exposure to bone marrow (BM) cells, blocks the toxin's effect, and a restoration of colony formation is achieved. A dose-dependent inhibition of CSF-induced colony formation is also observed in the presence of choleragenoid, the B subunit of the toxin which binds to the specific receptor on the cell surface, but is biologically inactive. Incubation of BM cells with CT prior to cloning in soft agar cultures supplemented with CSF inhibits clonal proliferation of CFUC, whereas a brief in vitro exposure of BM cells to CSF prior to CT protects the cells against subsequent CT inhibition. The ability of CT to inhibit the CSF-induced clonal proliferation of CFUC and the effectiveness of CSF in reducing and even counteracting CT inhibition suggests that CT, by binding to BM cells through the B subunit, might either directly or indirectly interfere with the stimulatory activity of CSF upon its target cells.     

Role of type 1 T helper cells in the resolution of acute Streptococcus pneumoniae sinusitis: a mouse model

BACKGROUND: We examined the importance of the adaptive and innate immune responses in the resolution of an acute bacterial sinus infection in mice. Methods: Recombinase-activating gene knockout (RAG-1(-/-)) (no lymphocytes) and C57BL/6 (wild-type) mice were infected with Streptococcus pneumoniae. For determination of the cell type involved, lymphocytes from mice were adoptively transferred into RAG-1(-/-), C57BL/6 (all lymphocytes), B cell-deficient, and T cell-deficient mice. The degree of infection and inflammation was determined by quantification of S. pneumoniae from nasal lavage and analysis of sinus tissue, respectively. RESULTS: In C57BL/6 mice, both the infection and inflammation resolved in 21 days, whereas neither resolved in RAG-1(-/-) mice. When C57BL/6 lymphocytes were adoptively transferred into RAG-1(-/-) mice, resolution of the infection and inflammation occurred. Mice without B cells were able to clear the infection, whereas mice without T cells could not clear it. In vitro stimulation of the draining lymph nodes of the infected mice by use of heat-killed S. pneumoniae led to the production of interferon (IFN)- gamma. Flow-cytometric analysis of lymphocytes obtained from sinus mucosa and draining lymph nodes showed an increase in the number of type 1 T helper cell-like cells over that in control mice. CONCLUSIONS: RAG-1(-/-) mice with innate immunity but no lymphocytes contain--but cannot clear--a bacterial sinus infection. Lymphocytes transferred to RAG-1(-/-) mice clear the infection. The sinus mucosa and draining lymph nodes show an increase in T cells generating IFN- gamma. These data demonstrate that T cells are essential in clearing an acute S. pneumoniae bacterial sinus infection.     

Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors

Two different types of cells in the peritoneal cavity of mice produce mast cell colonies in methylcellulose. "Large" mast cell colonies are produced by bone marrow-derived precursors resembling lymphoid cells by light microscopy (L-CFU-Mast), whereas "medium" and "small" mast cell colonies are produced by morphologically identifiable mast cells (M-CFU- Mast and S-CFU-Mast, respectively). In the present study we eradicated peritoneal mast cells by intraperitoneal (IP) injection of distilled water. The regeneration process was investigated to clarify the relationship between L-CFU-Mast, M-CFU-Mast, and S-CFU-Mast. After injection of distilled water, M-CFU-Mast and S-CFU-Mast disappeared, but L-CFU-Mast increased, and then M-CFU-Mast and S-CFU-Mast appeared, suggesting the presence of a hierarchic relationship. When purified peritoneal mast cells were injected two days after the water injection, the L-CFU-Mast did not increase. In the peritoneal cavity of WBB6F1-+/+ mice that had been lethally irradiated and rescued by bone marrow cells of C57BL/6-bgJ/bgJ (beige, Chediak-Higashi syndrome) mice, L-CFU-Mast were of bgJ/bgJ type, but M-CFU-Mast and S-CFU-Mast were of +/+ type. The injection of distilled water to the radiation chimeras resulted in the development of bgJ/bgJ-type M-CFU-Mast and then S-CFU-Mast. The presence of mast cells appeared to suppress the recruitment of L-CFU- Mast from the bloodstream and to inhibit the differentiation of L-CFU- Mast to M-CFU-Mast.     

Extensive proliferation of subsequently injected marrow cells in parental-to-F1 hematopoietic chimeras that restored normal stem cell concentration after initial transplantation

We investigated the fate of donor stem cells that were injected into hosts with a normal concentration of spleen colony-forming unit (CFU-S). Radiation chimeras were used as hosts. When CFU-S concentration in the marrow and spleen recovered to preirradiation levels after the initial bone marrow transplantation, the subsequent transplantation was done without reirradiation. Giant granules of beige C57B1/6 (bg) mice were used as a marker and proliferation and differentiation of the stem cells of the subsequent donor origin were evaluated by measuring the proportion of neutrophils with giant granules. No beige-type neutrophils were detectable at week 24 after transplantation of 5 X 10(7) marrow cells from bg mice to intact (WB X C57B1/6)F1 (F1) mice, which were used as control recipients. In contrast, transplantation of 5 X 10(7) marrow cells to radiation chimeras resulted in the appearance of neutrophils of second-donor origin. The proportion of beige-type neutrophils was 12% at week 24 after transplantation of bg marrow cells to F1-to-F1 (syngenic) or C57B1/6-+/+-to-bg (B6-to-bg) (congenic) chimeras; the proportion of beige-type neutrophils was 43% when bg marrow cells were transplanted to B6-to-F1 semiallogenic chimeras; the proportion of normal-type neutrophils was 82% when F1 marrow cells were injected to bg-to-F1 semiallogenic chimeras. Thus, the interaction of the host hematopoietic microenvironment with the stem cells of the initial donor as well as with the stem cells of the second donor seems to influence the proliferation and differentiation of the latter stem cells.     

Clonal analysis of the anti-DNA repertoire of murine B lymphocytes.

The present studies characterize at the clonal level the repertoire of lipopolysaccharide-responsive murine B lymphocytes committed to the production of antibodies reactive with denatured DNA. This repertoire is vast in normal mice as 1-5% of total mitogen-induced antibody-forming cell clones secreted denatured DNA-reactive antibodies when the splenocyte donors were CBA (Ighj), BALB/c (Igha), C57BL/6 (Ighb), CBA nu/nu, and C57BL/6 nu/nu athymic mice. The autoimmune NZB (Ighe) strain did not display elevated proportions of anti-denatured DNA antibody-forming cell precursors. Cross-reactions shown by CBA anti-denatured DNA antibodies suggest that many antibodies might derive significant binding energy from interaction with the bases or similar hydrophobic moieties. Cross-reactions with other tested polynucleotides were frequent, but cross-reactions with phospholipids and phosphocholine were undetectable. Most anti-DNA antibodies bound preferentially or exclusively to single-stranded denatured DNA as compared to double-stranded native DNA. The frequency of anti-denatured DNA antibody-forming cell precursors among CBA peritoneal cells was not elevated. Fluorescence-activated cell sorter-selected Ly-1-positive NZB splenic B cells were not enriched, and Ly-1 negative B cells were not depleted of anti-DNA antibody-forming cell precursors. These results show that antibody-forming cell precursors specific for denatured DNA are not restricted to the Ly-1 positive B-cell subset.