IL-15-deficient mice develop enhanced allergic responses to airway allergen exposure.

BACKGROUND: Interleukin-15 is a pleiotropic cytokine that is critical for the development and survival of multiple haematopoietic lineages. Mice lacking IL-15 have selective defects in populations of several pro-allergic immune cells including natural killer (NK) cells, NKT cells, and memory CD8+ T cells. We therefore hypothesized that IL-15-/- mice will have reduced inflammatory responses during the development of allergic airway disease (AAD). OBJECTIVE: To determine whether IL-15-/- mice have attenuated allergic responses in a mouse model of AAD. METHODS: C57BL/6 wild-type (WT) and IL-15-/- mice were sensitized and challenged with ovalbumin (OVA), and the development of AAD was ascertained by examining changes in airway inflammatory responses, Th2 responses, and lung histopathology. RESULTS: Here, we report that IL-15-/- mice developed enhanced allergic responses in an OVA-induced model of AAD. In the absence of IL-15, OVA-challenged mice exhibited enhanced bronchial eosinophilic inflammation, elevated IL-13 production, and severe lung histopathology in comparison with WT mice. In addition, increased numbers of CD4+ T and B cells in the spleens and bronchoalveolar lavage (BAL) were also observed. Examination of OVA-challenged IL-15Rα-/- animals revealed a similar phenotype resulting in enhanced airway eosinophilia compared to WT mice. Adoptive transfer of splenic CD8+ T cells from OVA-sensitized WT mice suppressed the enhancement of eosinophilia in IL-15-/- animals to levels observed in WT mice, but had no further effects. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that mice with an endogenous IL-15 deficiency are susceptible to the development of severe, enhanced Th2-mediated AAD, which can be regulated by CD8+ T cells. Furthermore, the development of disease as well as allergen-specific Th2 responses occurs despite deficiencies in several IL-15-dependent cell types including NK, NKT, and γδ T cells, suggesting that these cells or their subsets are dispensable for the induction of AAD in IL-15-deficient mice.

Pro-inflammatory role of natural killer cells in the development of allergic airway disease.

BACKGROUND: Natural Killer (NK) cells have been implicated in the development of allergic airway inflammation. However, the in vivo role of NK cells has not been firmly established due to the lack of animal models with selective deficiencies in NK cells. OBJECTIVE: To determine the specific contribution of NK cells in a murine model of allergic airway disease (AAD). METHODS: The role of NK cells in AAD was studied using NK-deficient (NKD) mice, perforin(-/-) mice, and mice depleted of Ly49A/D/G(+) NK cell subsets in an ovalbumin-induced model of allergic airway disease (OVA-AAD). RESULTS: Induction of OVA-AAD in C57BL/6 wild-type (WT) mice resulted in the expansion of airway NK cells and the development of pronounced airway eosinophilia. In the absence of NK cells or specific subsets of NK cells, either in NKD mice, or after the depletion of Ly49A/D/G(+) NK cells, the development of OVA-AAD was significantly impaired as seen by decreased airway inflammation and eosinophilia, decreased secretion of the Th2 cytokines IL-4, IL-5 and IL-13 and diminished OVA-specific antibody production. Furthermore, while OVA-exposure induced a dramatic expansion of dendritic cells (DCs) in WT mice, their induction was significantly attenuated in NKD mice. Development of OVA-AAD in perforin(-/-) mice suggested that the proinflammatory role of NK cells is not dependent on perforin-mediated cytotoxicity. Lastly, induction of allergic disease by OVA-specific CD4 T cells from WT but not NK-depleted or NKD mice in RAG(-/-) recipients, demonstrates that NK cells are essential for T cell priming. CONCLUSIONS AND CLINICAL RELEVANCE: Our data demonstrate that conventional NK cells play an important and distinct role in the development of AAD. The presence of activated NK cells has been noted in patients with asthma. Understanding the mechanisms by which NK cells regulate allergic disease is therefore an important component of treatment approaches.

Defining a visual marker of osteoprogenitor cells within the periodontium.

BACKGROUND AND OBJECTIVE: Cells with osteoprogenitor potential are present within periodontal tissues during development and in postnatal life. To identify an osteoprogenitor population, this study utilized a transgenic model in which an alpha-smooth muscle actin (alphaSMA) promoter directed green fluorescent protein (GFP) expression. MATERIAL AND METHODS: Observation of GFP expression was complemented with analysis of osteogenic differentiation by determining the expression of RNA of bone markers, by histochemical staining for alkaline phosphatase and by the detection of mineralized nodules using xylenol orange. Flow cytometry was utilized to determine the proliferative potential and cell-surface phenotype of cultured alphaSMA-positive cells. RESULTS: alphaSMA-GFP expression was detected within the dental follicle and in the apical region of the root (i.e. areas rich in vascularization) but not in mature bone. alphaSMA-GFP expression was observed during the early stages of primary cultures derived from the dental follicle and periodontal ligament and was diminished in areas undergoing mineralization. Intense alkaline phosphatase activity and the presence of mineralized nodules was observed 2 wk after osteogenic induction. Consequently, the expression of bone sialoprotein, osteocalcin and dentin matrix protein-1 was increased. Flow cytometry revealed that in vitro expansion enriched for an alphaSMA-GFP-positive population in which 55-65% of cells expressed the cell-surface markers Thy1(+) and Sca1(+). The alphaSMA-GFP-positive population exhibited high proliferative and osteogenic potentials when compared with an alphaSMA-GFP-negative population. CONCLUSION: Our data indicate that the alphaSMA promoter can be used to identify a population of osteoprogenitor cells residing within the dental follicle and periodontal ligament that can differentiate into mature osteoblasts.

Conditional ablation of the osteoblast lineage in Col2.3deltatk transgenic mice.

Two transgenic mouse lines were generated with a DNA construct bearing a 2.3-kilobase (kb) fragment of the rat alpha1 type I collagen promoter driving a truncated form of the herpes thymidine kinase gene (Col2.3Atk). Expression of the transgene was found in osteoblasts coincident with other genetic markers of early osteoblast differentiation. Mice treated with ganciclovir (GCV) for 16 days displayed extensive destruction of the bone lining cells and decreased osteoclast number. In addition, a dramatic decrease in bone marrow elements was observed, which was more severe in the primary spongiosum and marrow adjacent to the diaphyseal endosteal bone. Immunostaining for transgene expression within the bone marrow was negative and marrow stromal cell cultures developed normally in the presence of GCV until the point of early osteoblast differentiation. Our findings suggest that the early differentiating osteoblasts are necessary for the maintenance of osteoclasts and hematopoiesis. Termination of GCV treatment produced an exaggerated response of new bone formation in cortical and trabecular bone. The Col2.3deltatk mouse should be a useful model to define the interrelation between bone and marrow elements as well as a model to analyze the molecular and cellular events associated with a defined wave of osteogenesis on termination of GCV treatment.

Estradiol rapidly inhibits osteoclastogenesis and RANKL expression in bone marrow cultures in postmenopausal women: a pilot study.

UNLABELLED: We examined RANKL expression and OCL formation in cultured bone marrow cells from eight postmenopausal women in response to E(2). E(2) treatment inhibited the ability of hematopoietic cells to form OCLs in response to RANKL, and decreased RANKL production. These changes are likely involved in the ability of E(2) to influence the development of osteoporosis. INTRODUCTION: Estrogen (E(2)) deficiency at menopause increases osteoclast (OCL) formation and bone resorption, predisposing women to osteoporosis. We examined receptor activator of NF-kappa B-ligand (RANKL) expression and in vitro OCL formation in cultured bone marrow cells from eight postmenopausal women before and after 3 weeks of E(2) therapy and three untreated premenopausal women. METHODS: TRAP staining and resorption pit assay determined OCL number and function. Flow cytometry measured the distribution of marrow cell types and expression of RANKL in the macrophage-enriched fraction (R1) and a lymphocyte-enriched fraction (R2). RESULTS: RANKL (3-100 ng/ml) produced a dose-dependent increase in in vitro OC formation and E(2) therapy significantly (p < 0.01) inhibited OCL formation by 33 to 50%. A small proportion of marrow cells bound anti- RANKL Ab (0.2-4.3%). There was no effect of E(2) on the percentage of cells binding the anti-RANKL Ab in the R1 fraction. In the R2 fraction E(2) treatment decreased the percentage of cells binding anti-RANKL Ab by 68 +/- 9% (p < 0.01). CONCLUSION: Three weeks of E(2) treatment had a dual action. It inhibited the ability of hematopoietic cells to form OCLs in response to RANKL, and decreased the production of RANKL in cells of the bone marrow. The observed changes in the osteoclastic potential of bone marrow cells are likely involved in the ability of E(2) to regulate bone mass and influence the development of osteoporosis.

Hematopoietic stem cells are not direct cytotoxic targets of natural killer cells.

Bone marrow (BM) transplants from one individual to an irradiated histoincompatible individual of the same species are rejected. In mice, the primary host barrier cells that recognize bone marrow grafts bearing hematopoietic histocompatibility antigens bear surface markers of natural killer (NK) lymphocytes. Because of the innate ability of NK cells to kill susceptible targets, it has been proposed that the cytotoxic bone marrow graft rejection. To test this hypothesis, we purified hematopoietic stem cells from mice and incubated them with purified populations of actively cytotoxic allogeneic and semisyngeneic NK cells, followed by analysis of the ability of the treated hematopoietic stem cells (HSCs) to rescue lethally irradiated syngeneic animals. Such rescue was unimpaired. Also, HSC allografts were transplanted into transgenic mice deficient in NK and killer T-cell cytotoxicity generated by expressing diphtheria toxin A chain under the control of granzyme A promoter. Allogeneic HSCs were susceptible to allogeneic restriction in these mice, implying that the effector functions of NK marker-positive cells do not require NK cell cytotoxicity.

T15 PC-binding monoclonal antibodies retain specificity when they switch from IgM to IgG.

The expression of some Ag-binding sites and their associated H and L chain V region genes are often dominated by one or another of the IgG subclasses. This is true of T15 anti-phosphorylcholine (PC) response in which the dominant T15 H (V1, DFL11.6 JH1) and L (VK22, JK5) chain V regions are seldom found associated with the gamma 2b or gamma 2a C regions in the circulation of BALB/c mice. In the present study we describe the characterization of gamma 2b, gamma 2a, and gamma 1 anti-PC antibodies obtained from IgM-producing hybridomas by Ig switching in culture. All switch variants retained the parental T15 idiotype, PC binding, and fine specificity. We thus conclude that the apparent in vivo restriction on the expressed T15 PC binding gamma 2 antibodies is not due to a conformational limitation on the expression of the anti-PC T15 binding site. Further, these studies confirm that in vitro switching can be used to generate mAb that are not readily available in vivo.

Amino terminal sequence of heavy and light chains from ratfish immunoglobulin.

The ratfish, Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (Mr 960,000), composed of heavy (Mr 71,000), light (Mr 22,500), and J (Mr 16,000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark, Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.

Bcl-2 prevents death of factor-deprived cells but fails to prevent apoptosis in targets of cell mediated killing.

'Programmed cell death' has been used to describe the death of cells killed by cytotoxic T cells or growth factor deprivation. Although bcl-2 can prevent death of cells deprived of growth factor, it failed to protect cells against T cell killing. In spite of bcl-2 expression, the DNA of targeted cells was degraded into nucleosome-sized fragments. Therefore the early steps in apoptosis induced by factor deprivation differ from those triggered by cytotoxic T cells, although they share a common final pathway featuring degradation of the DNA and loss of cytoplasmic membrane integrity.

Transgenic mice carrying the diphtheria toxin A chain gene under the control of the granzyme A promoter: expected depletion of cytotoxic cells and unexpected depletion of CD8 T cells.

We have generated transgenic mice bearing the diphtheria toxin A chain (DTA) gene under the control of granzyme A (GrA) promoter sequences (GrA-DTA). GrA is expressed in activated cytotoxic cells but not in their immediate progenitors. These GrA-DTA mice are deficient in cytotoxic functions, indicating that most cytotoxic cells express GrA in vivo. Surprisingly, one founder strain containing a multicopy GrA-DTA insert show a marked and selective deficiency in CD8+ cells in peripheral lymphoid organs. This depletion was not observed in thymus, where the distribution of CD4+ and CD8+ cells is normal. Moreover, the emigration of T cells from thymus is normal, indicating that the depletion occurs in the periphery. GrA-DTA mice should be useful as models to dissect the role of cytotoxic cells in immune responses and as recipients of normal and neoplastic hematopoietic cells. The selective depletion of CD8+ cells in one founder strain could have implications for postthymic T-cell development.

The molecular and biochemical characterization of mutant monoclonal antibodies with increased antigen binding.

Mutant mAb with increased Ag binding were generated from a hybridoma cell line, 36-65, that secretes an IgG1,kappa anti-p-azophenylarsonate-(Ars) specific antibody. The mutant antibodies were identified using an Ars-specific ELISA and sib selection so that approximately 10(6) cells could be analyzed. The ELISA used as Ag a low ratio of Ars coupled to BSA and was set up so that only those antibodies that had higher binding than the parent would be detected. Seven mutant producing cell lines were isolated from five independent clones of 36-65. The mutant antibodies bind Ag 20 to more than 200-fold better than the parent and have wild type V region sequences. All have C region mutations that result in an increased avidity. At least five different genetic events are responsible for the C region mutations.

Clonal variants of hybridoma cells that switch isotype at a high frequency.

As B cells differentiate under the influence of antigen and T cells, they frequently switch from the expression of IgM antibody to the expression of other isotypes. This is accomplished by rearranging the expressed variable region gene to downstream constant region genes and deleting the intervening sequences. Some B-cell lines that represent early stages in development switch constitutively in culture at frequencies that approach those of lipopolysaccharide- or lymphokine-stimulated normal B cells. Hybridoma cells represent a later stage of development and rarely switch in culture. In contrast to early B-cell lines, hybridomas produce large amounts of immunoglobulin, and single cells can be assayed easily for the expression of new isotypes. We have used the ELISA spot assay and fluctuation analysis to determine the rate of switching of two hybridoma cell lines. By identifying subclones that switched more frequently, we have progressively enriched for cells that switch spontaneously at higher rates. These cells, like normal cells, switch by rearrangement and deletion, and the frequency of switched cells in some of the clones is comparable to that which has been observed in less differentiated B-cell lines and in normal B cells.

Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are believed to play a critical role in the sustained repopulation of all blood cells after bone marrow transplantation (BMT). However, understanding the role of HSCs versus other hematopoietic cells in the quantitative reconstitution of various blood cell types has awaited methods to isolate HSCs. A candidate population of mouse HSCs, Thy-1.1lo Lin-Sca-1+ cells, was isolated several years ago and, recently, this population has been shown to be the only population of BM cells that contains HSCs in C57BL/Ka-Thy-1.1 mice. As few as 100 of these cells can radioprotect 95% to 100% of irradiated mice, resulting long-term multilineage reconstitution. In this study, we examined the reconstitution potential of irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ BM cells. Donor-derived peripheral blood (PB) white blood cells were detected as early as day 9 or 10 when 100 to 1,000 Thy-1.1lo Lin-Sca-1+ cells were used, with minor dose-dependent differences. The reappearance of platelets by day 14 and thereafter was also seen at all HSC doses (100 to 1,000 cells), with a slight dose-dependence. All studied HSC doses also allowed RBC levels to recover, although at the 100 cell dose a delay in hematocrit recovery was observed at day 14. When irradiated mice were transplanted with 500 Thy-1.1lo Lin-Sca-1+ cells compared with 1 x 10(6) BM cells (the equivalent amount of cells that contain 500 Thy-1.1lo Lin-Sca-1+ cells as well as progenitor and mature cells), very little difference in the kinetics of recovery of PB, white blood cells, platelets, and hematocrit was observed. Surprisingly, even when 200 Thy1.1lo Lin-Sca-1+ cells were mixed with 4 x 10(5) Sca-1- BM cells in a competitive repopulation assay, most of the early (days 11 and 14) PB myeloid cells were derived from the HSC genotype, indicating the superiority of the Thy-1.1lo Lin-Sca-1+ cells over Sca-1- cells even in the early phases of myeloid reconstitution. Within the Thy-1.1lo Lin-Sca-1+ population, the Rhodamine 123 (Rh123)hi subset dominates in PB myeloid reconstitution at 10 to 14 days, only to be overtaken by the Rh123lo subset at 3 weeks and thereafter. These findings indicate that HSCs can account for the early phase of hematopoietic recovery, as well as sustained hematopoiesis, and raise questions about the role of non-HSC BM populations in the setting of BMT.

In vivo natural killer cell activities revealed by natural killer cell-deficient mice.

Studies of natural killer (NK) cell function in vivo have been challenging primarily due to the lack of animal models in which NK cells are genetically and selectively deficient. Here, we describe a transgenic mouse with defective natural killing and selective deficiency in NK1.1(+) CD3(-) cells. Despite functionally normal B, T, and NK/T cells, transgenic mice displayed impaired acute in vivo rejection of tumor cells. Adoptive transfer experiments confirmed that NK1.1(+) CD3(-) cells were responsible for acute tumor rejection, establishing the relationship of NK1.1(+) CD3(-) cells to NK cells. Additional studies provided evidence that (i) NK cells play an important role in suppressing tumor metastasis and outgrowth; (ii) NK cells are major producers of IFNgamma in response to bacterial endotoxin but not to interleukin-12, and; (iii) NK cells are not essential for humoral responses to T cell-independent type 2 antigen or the generalized Shwartzman reaction, both of which were previously proposed to involve NK cells.

Studies on the somatic instability of immunoglobulin genes in vivo and in cultured cells.

We have examined the molecular mechanism and impact of somatic diversification on the T15 heavy chain variable region gene in vivo and in vitro. Somatic point mutation appears to be responsible for the changes we have observed in both hybridomas from early and late in the immune response and in the S107 myeloma cell line in culture. By identifying S107 mutants with decreases in antigen binding, we have shown that a single point mutation can cause the loss of binding to the eliciting antigen and the acquisition of binding to another antigen. Furthermore, in this case a point mutation of the T15 heavy chain variable region gene caused the conversion of an important protective antibody to an autoantibody. While the S107 cell line frequently generates both constant and variable region mutants, hybridomas appear to have relatively stable variable region genes and unstable constant region genes which in some cases result in mutants with increased binding.

From stem cells to lymphocytes: biology and transplantation.

We review the development of the hematopoietic system, focusing on the transition from hematopoietic stem cells (HSCs) to T cells. This includes the isolation of HSCs, and recent progress in understanding their ontogeny, homing properties, and differentiation. HSC transplantation is reviewed, including the kinetics of reconstitution, engraftment across histocompatibility barriers, the facilitation of allogeneic engraftment, and the mechanisms of graft rejection. We describe progress in understanding T-cell development in the bone marrow and thymus as well as the establishment of lymph nodes. Finally, the role of bcl-2 in regulating homeostasis in the hematopoietic system is discussed.