Elastase and cathepsin G activities are present in immature bone marrow neutrophils and absent in late marrow and circulating neutrophils of beige (Chediak-Higashi) mice.

Elicited peritoneal neutrophils of beige (Chediak-Higashi) mice essentially lack activities of the neutral serine proteinases elastase and cathepsin G, which may explain the increased susceptibility to infection of beige mice and Chediak-Higashi patients. We have examined neutrophils of beige mice at earlier points in their development to determine if the proteinase genes are never expressed or whether they are expressed and then lost during neutrophil maturation. Surprisingly, bone marrow of beige mice had significant elastase and cathepsin G activity (approximately 60% of normal). The results of several experiments indicate that neutrophils were the sole source of elastase and cathepsin G in bone marrow. Neutral proteinase activity was readily demonstrable by histochemical procedures in beige marrow neutrophil precursors up to and including the metamyelocyte stage. However, mature neutrophils of beige marrow had greatly decreased activity. Also mature neutrophils (PMNs) of the peripheral circulation, like peritoneal neutrophils, had very low elastase and cathepsin C activities. Thus we conclude that beige neutrophil precursors express neutral proteinase activity, which is largely and irreversibly depleted by the time they fully mature in marrow.

Lymphoid cell dependence of eosinophil response to antigen.

Quantitative determinations were made of the capacity of isogenic bone marrow, spleen, and thymic cells from primed and/or nonprimed mice to repopulate the hemopoietic tissues and to mount an inflammatory and antibody response to specific antigen (tetanus toxoid) in heavily irradiated and reconstituted recipients. Spleen cells from primed mice but not from normal mice had the capacity to adoptively transfer an anamnestic antitoxin titer in irradiated animals in the absence of transplanted bone marrow cells, and during retarded myeloid regeneration. Spleen cells alone or bone marrow cells alone produced an insignificant and a moderate peritoneal eosinophil response, respectively, to antigen. In the presence of bone marrow cells, normal spleen cells augment the capacity of recipient animals to mount an eosinophil response to antigen. A much greater augmentation occurs in animals reconstituted with splenic or thymic cells from primed animals. The increase in antitoxin titers appears to be independent of the response of eosinophils since: (a) marked accumulation of eosinophils can occur in animals with no measurable humoral antitoxin, and (b) high antitoxin titers can occur in animals which do not have marked eosinophil responses. It is suggested that a thymic-derived or thymic-dependent mononuclear cell population is necessary for optimal eosinophil response to antigen. The neutrophil and mononuclear cell responses to antigen are determined by different mechanisms from those which determine the eosinophil response. These studies together with earlier findings strongly indicate that the eosinophil granulocytes play a role in the immune response to antigen.

Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma.

We have generated transgenic mice that constitutively express murine interleukin (IL)-5 in the lung epithelium. Airway expression of this cytokine resulted in a dramatic accumulation of peribronchial eosinophils and striking pathologic changes including the expansion of bronchus-associated lymphoid tissue (BALT), goblet cell hyperplasia, epithelial hypertrophy, and focal collagen deposition. These changes were also accompanied by eosinophil infiltration of the airway lumen. In addition, transgenic animals displayed airway hyperresponsiveness to methacholine in the absence of aerosolized antigen challenge. These findings demonstrate that lung-specific IL-5 expression can induce pathologic changes characteristic of asthma and may provide useful models to evaluate the efficacy of potential respiratory disease therapies or pharmaceuticals.

Eosinophils in health and disease: the LIAR hypothesis.

Discussions of eosinophils are often descriptions of end-stage effector cells with destructive capabilities mediated predominantly by released cytotoxic cationic granule proteins. Moreover, eosinophils in the medical literature are invariably associated with the pathologies linked with helminth infections or allergic diseases such as asthma. This has led to an almost fatalist view of eosinophil effector functions and associated therapeutic strategies targeting these cells that would make even William of Ockham proud - eosinophil effector functions have physiological consequences that increase patient morbidity/mortality and 'the only good eosinophils are dead eosinophils'. Unfortunately, the strengths of dogmas are also their greatest weaknesses. Namely, while the repetitive proclamation of dogmatic concepts by authoritative sources (i.e. reviews, meeting proceedings, textbooks, etc.) builds consensus within the medical community and lower the entropies surrounding difficult issues, they often ignore not easily explained details and place diminished importance on alternative hypotheses. The goal of this perspective is twofold: (i) we will review recent observations regarding eosinophils and their activities as well as reinterpret earlier data as part of the synthesis of a new paradigm. In this paradigm, we hypothesize that eosinophils accumulate at unique sites in response to cell turnover or in response to local stem cell activity(ies). We further suggest that this accumulation is part of one or more mechanisms regulating tissue homeostasis. Specifically, instead of immune cells exclusively mediating innate host defence, we suggest that accumulating tissue eosinophils are actually regulators of Local Immunity And/or Remodeling/Repair in both health and disease - the LIAR hypothesis; (ii) we want to be inflammatory (pun intended!) and challenge the currently common perspective of eosinophils as destructive end-stage effector cells. Our hope is to create more questions than we answer and provoke everyone to spend countless hours simply to prove us wrong!

Expression of IL-5 alters bone metabolism and induces ossification of the spleen in transgenic mice.

We have developed a transgenic mouse line, NJ.1638, which expresses high levels of IL-5 from T cells, with profound hematological consequences. Eosinophils comprise more than 60% of circulating white blood cells in these animals, with the total peripheral white blood cell counts increasing more than 40-fold relative to wild-type littermates. This extraordinary proliferative capacity is sustained by expanded sites of extramedullary hematopoiesis and is accompanied by multifocal, ectopic bone formation in the spleen. Histology of the splenic nodules revealed the presence of osteoid matrices and osteocytes trapped within mineralized trabecular plates. In addition, polarized light microscopy of calcified tissue sections revealed both woven bone and areas of organized lamellar bone. Morphometric assessments demonstrated that both the growth and mineralization of splenic bone occurred at rates nearly an order of magnitude higher than in skeletal bone. Skeletal bone metabolic parameters were also perturbed. We also observed heterotopic ossification of the spleen and perturbation of skeletal bone homeostasis following adoptive engraftment of transgenic marrow to wild-type recipients. These data suggest that IL-5 overexpression mediates bone formation through the mobilization of marrow-derived osteogenic progenitors and/or the inhibition of recruited osteoclasts.

In vivo distinction between a target cell for Friend virus (FVP) and murine hematopoietic stem cells.

Busulphan (BU) treatment of DBA/2 mice with hypertransfusion (HT)-induced polycythemia resulted in an ablation of detectable hematopoietic stem cells (CFUS) in pooled marrow from the long bones. Daily injections of erythropoietin (EP) stimulated an EP-responsive population of cells in the absence of detectable CFUS. Mice treated with BU and EP and having HT-induced polycythemia were inoculated with the polycythemia-inducing strain of Friend virus (FVP) and determinations were made for the presence of tumor colony-forming units (tCFU). No change in CFUS/10(6) bone marrow cells was detected as a result of EP treatment. However, tCFU were increased more than 100-fold in HT-BU-EP-treated mice compared with saline-treated controls. The demonstration of tCFU in mice in which CFUS were not detectable indicated that this leukemogenic effect of FVP could occur in the absence of the pluripotent stem cell. Furthermore, the increased numbers of this FVP target cell in the EP-stimulated, BU-treated mice with HT-induced polycythemia supported the model licating the target for this effect in the EP-responsive cell population.

Friend virus-induced inhibition of eosinophil granulocyte exudation in mice.

Exudation of eosinophil polymorphonuclear leukocytes (E-PMN) in response to tetanus toxoid (TT) was studied in DBA/2HaD mice with erythroleukemia induced by Friend virus (FV). The inhibition of exudation that developed was independent of increased levels of serum corticosteroids and occurred in surgically adrenalectomized mice. Thus it was independent of steroid-induced effects on E-PMN. The accumulation of neutrophil polymorphonuclear leukocytes (N-PMN) in TT-induced exudates was unaltered. Furthermore, N-PMN exudation in response to other inflammatory stimuli was similarly unimpaired in virus-infected mice, which confirmed the specificity of the inhibition for E-PMN. The virus entity in the FV complex responsible for the effect was not identified. Friend murine leukemia virus, the indigenous helper virus for the defective spleen focus-forming virus, alone, was incapable of inducing the inhibition. It is possible that the lack of participation of E-PMN in TT-induced immune inflammatory exudates in FV-infected mice reflects an unresponsiveness that contributes to the development and progression of leukemia in FV-infected mice.

Erythropoietin responsiveness in vivo of Friend virus target cells for transformation and virus replication.

Techniques that allow the selective stimulation of the erythropoietin-responsive cell population in mice with suppressed multipotential hemopoietic stem cells were used to identify (1) the in vivo target cell transformed by Friend virus (FV) into a tumor colony-forming unit and (2) a target cell for FV replication in vivo. Plethoric mice with busulphan-induced reductions in stem cell populations (characterized as colony-forming units) and stimulated erythropoietin-responsive cell compartments were given FV; control groups, not receiving erythropoietin, also received FV. A comparison of the number of target cells transformed in each group provided evidence identifying the ERC as the in vivo compartment in which the target cell detected by tumor colony formation resides. Differences in plasma virus titers revealed that the erythropoietin-responsive cell is also predominantly responsible for the production of FV as measured by focus-forming activity.

Murine eosinophil granulocytes bind the murine macrophage-monocyte specific monoclonal antibody F4/80.

Studies designed to identify a panel of monoclonal antibodies useful for the separation of murine eosinophil myeloid progenitors revealed that F4/80, an antigen heretofore thought to be expressed only by murine monocyte/macrophage lineage cells, was expressed by eosinophil granulocytes. Eosinophils from several strains of mice stained positively with specific antibody for the epitope. A novel pathway for myeloid differentiation is proposed in which neutrophil progenitors exit a common lineage prior to a common progenitor of monocytes and eosinophils. Moreover, the results demonstrate that binding of anti-F4/80 can no longer be viewed as exclusive for mononuclear phagocytes.

Murine eosinophilopoiesis: dose-dependent response to soluble antigens of Schistosoma mansoni.

Initial and (or) subsequent exposures of animals to selected antigens result in impressive accumulations of eosinophil granulocytes at the site of challenge. When it has been studied, the exudation is accompanied by an increase in medullary eosinophilopoiesis. The medullary response is proportional to the amount of antigen used to induce the exudate as shown by responses to single challenge injections with graded doses of a soluble antigen preparation from schistosome eggs (SEA). Attempts were made to mimic the continuous antigen challenge of chronic schistosomiasis by use of osmotic minipumps and intermittent injections with SEA. These did not result in prolonged increases of marrow eosinophilopoiesis for reasons which remain unclear.

Murine eosinophil granulocyte proliferation kinetics in acute and chronic eosinophilia.

Eosinophil proliferation kinetics have been determined by examining fraction-labeled mitoses (FLM) curves in femoral marrow of mice stimulated for eosinophil production. Mice with marrow eosinophilopoiesis increased as a result of infection with the parasite Schistosoma mansoni exhibited eosinophil cycle durations of 22 h. This time is not different from normal rates of murine eosinophil cell cycles. Acutely stimulated mice, however, with a rapid eosinophilia initiated by rechallenge with a soluble schistosome egg antigen preparation, had marrow eosinophil proliferative cycles of about 10 h. The long duration of eosinophil cell cycles in mice with an extensive commitment to the production of large numbers of eosinophil granulocytes suggests that several mechanisms regulate expansions of eosinophil granulocytopoiesis in vivo by hemopoietic marrow.

Progenitor cell defect correctable by bone marrow transplantation in five independent mouse models of platelet storage pool deficiency.

Two human platelet storage pool deficiencies (SPD), Hermansky-Pudlak syndrome and Chediak-Higashi syndrome, are recessively inherited and characterized by hypopigmentation, prolonged bleeding, and normal platelet numbers accompanied by a reduction of platelet dense granules. Seven independent and unique mouse pigment mutations regulated by separate genes have been proposed as animal models for SPD. Mice homozygous for the recessive mutations have diluted pigmentation, prolonged bleeding times, normal platelet concentrations, and reduced numbers of platelet dense granules. Reciprocal bone marrow transplantations were carried out between normal C57Bl/6J mice and five of these mutants, pearl, light ear, pale ear, ruby-eye, and maroon, to test whether the platelet defects are due to platelet progenitor cells or to humoral regulatory factors. Recipient mice were transplanted with marrow after 950-rad whole body irradiation. The prolonged bleeding time and low serotonin concentrations of the five mutants were converted to normal values after transplantation with normal marrow. Normal mice displayed characteristics of platelet SPD when transplanted with mutant marrow. This study demonstrates that in each of five independent mouse models the thrombopathy of SPD is due to a platelet progenitor cell defect correctable by bone marrow transplantation. These findings suggest that in severe cases human SPD may be amenable to treatment by bone marrow transplantation.

Stimulation of erythropoiesis in a grafted animal by mouse fetal liver culture media.

Erythropoietin given to polycythemic, irradiated mice receiving a bone marrow transplant restores the number of erythroid colonies produced. In this study mouse fetal liver culture media administered to polycythemic, irradiated mice also restored the number of erythroid colonies and erythropoiesis as measured by 59Fe incorporation. Thus, mouse fetal liver in culture produces a factor which resembles erythropoietin.

Effects of mixed chimeric bone marrow repopulation on platelet storage pool-associated bleeding defects in mouse mutants.

We have previously shown mouse platelet storage pool deficiency (SPD) to be associated with lesions at eight different genetic loci, each of which is sufficient to produce murine SPD. We have also shown that normal bleeding times and normal platelet functions are restored when mice with SPD are transplanted with marrow from normal mice. Conversely, when normal mice are transplanted with mutant marrow, they present symptoms of SPD. In order to determine the amount of normal platelets needed to prevent the prolonged bleeding times associated with SPD, we established stable mixed chimeric mice by transplanting various ratios of normal and mutant marrow into lethally irradiated host animals. The proportion of normal input marrow correlated well with the proportion of normal peripheral red blood cells and platelets determined in chimerae 100 days after transplantation using direct morphology and electrophoretic variants of glucose phosphate isomerase to identify normal and mutant cell populations. The proportions of normal input marrow were also reflected in the proportions of platelets with normal and mutant platelet morphology in the chimerae. This confirms that the platelet abnormality in SPD is intrinsic to the stem cell population from which the platelets are derived. When bleeding times were determined in the mixed chimeric mice, a surprisingly high percentage of normal platelets (greater than 50% and sometimes greater than 75%) were needed to stop bleeding. These results suggest that the mutant platelets in the mixed chimeric mice may interfere with normal platelet aggregation patterns. They also raise some important considerations in devising treatment for SPD. Bleeding episodes in human SPD are normally treated by platelet transfusion. The results suggest that, at least in some cases, transfusions may not be effective. Also, in future gene therapy of this disease, it is like that a functional gene will have to be present in greater than 50% of stem cells for therapy to be effective.