Janet North

Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.

Kinetics of cell infiltration and cytokine messenger RNA expression after intradermal challenge with allergen and tuberculin in the same atopic individuals

BACKGROUND Previous studies, in which one time point was used, have shown that cells infiltrating skin biopsy specimens taken during allergen-induced late-phase responses (LPR) had a TH2-like (interleukin-4 [IL]-4 and IL-5 mRNA+) cytokine profile, whereas in delayed-type hypersensitivity (DTH) there was a predominant TH1-type pattern. OBJECTIVE The study was designed to examine the kinetics of accumulation of inflammatory cells and cells expressing mRNA for TH2- or TH1-type cytokines in LPR and DTH elicited simultaneously in the same subjects. METHODS Immunocytochemistry (alkaline phosphatase anti-alkaline phosphatase technique) and in situ hybridization were used to analyze skin biopsy specimens taken during allergen-induced LPR. RESULTS In LPR elevated numbers of CD3+ and CD4+ cells, eosinophils, neutrophils, and IL-4 and IL-5 mRNA+ cells were detected as early as 1 hour after allergen challenge, with a peak at 6 hours, which was maintained for up to 96 hours. A small but significant delayed increase in macrophages, CD8+ and CD25+ cells, and IL-2 and interferon-gamma mRNA+ cells was also observed, but only at the 48-hour and 96-hour time points. In contrast, in DTH the numbers of CD3+, CD4+, and mRNA+ cells for IL-2 and interferon-gamma were not elevated until 24 hours after challenge and peaked at 48 hours after injection. At 48 hours there was an additional small but significant increase in IL-4 and IL-5 mRNA+ cells. For both LPR and DTH the kinetics of the increases in inflammatory cells and cytokine mRNA-expressing cells paralleled the clinical response. CONCLUSIONS In LPR accumulation of T cells and granulocytes, together with cells expressing mRNA encoding for TH2-type cytokines, is relatively rapid (i.e., within 1 to 6 hours), whereas in DTH the T cell/macrophage infiltration and appearance of cells expressing TH1-type cytokines are not apparent until 24 to 48 hours. In LPR there is a TH1-type (or possibly TH0) component at 48 to 96 hours, and in DTH there is an additional TH2/TH0 response.

Tumor-Primed Human Natural Killer Cells Lyse NK-Resistant Tumor Targets: Evidence of a Two-Stage Process in Resting NK Cell Activation

NK cells are defined as those cells that lyse tumor cells without priming. In this study, we show that the preincubation of resting human NK cells with the leukemia cell CTV-1 primes NK cells to lyse NK-resistant cell lines, primary leukemias, and solid tumors even when HLA-matched, allogeneic or autologous. The primed NK cells remained nonresponsive to HLA-C matched and mismatched normal mononuclear cells from multiple donors. CD69, a known NK trigger receptor, was shown to be the predominant trigger on the tumor-primed NK cells because lysis was blocked with the rCD69 protein. The lack of lytic activity against normal hemopoietic cells implied that the ligand for CD69 is tumor restricted, and this was confirmed by experiments using fluorochrome labeled rCD69. It has been recently shown that resting NK cells require prior stimulation with IL-2 before triggering by all known NK-triggering ligands. In this study, we show that a tumor cell can provide the NK priming signal independently of IL-2. These data provide evidence for two NK evasion strategies for tumor cells, namely the prevention of priming (type1 evasion) and failure to trigger (type 2 evasion). Most NK-resistant cell lines are type 1 and fail to prime resting NK cells but are lysed by IL-2-primed NK cells. In contrast, CTV-1 cells prime resting NK cells but fail to trigger (type 2), and coincubation with CTV-1 primes for triggering by type 1 NK-resistant tumor cells. These tumor-activated NK cells lyse a broad spectrum of tumor cells with a degree of specificity never previously reported.

Selective Apoptotic Killing of Malignant Hemopoietic Cells by Antibody-Targeted Delivery of an Amphipathic Peptide

The alpha-helical amphipathic peptide D-(KLAKLAK)2 is toxic to eukaryotic cells if internalized by a suitable targeting mechanism. We have targeted this peptide to malignant hemopoietic cells via conjugation to monoclonal antibodies, which recognize lineage-specific cell surface molecules. An anti-CD19/peptide conjugate efficiently killed 3/3 B lymphoid lines. However, an anti-CD33/peptide conjugate was cytotoxic to only one of three CD33-positive myeloid leukemia lines. The IC50 towards susceptible lines were in the low nanomolar range. Conjugates were highly selective and did not kill cells that did not express the appropriate cell surface cognate of the antibody moiety. Anti-CD19/peptide conjugates efficiently killed cells from patients with chronic lymphocytic leukemia but anti-CD33/peptide reagents were less effective against fresh acute myeloid leukemia cells. We therefore suggest that amphipathic peptides may be of value as targeted therapeutic agents for the treatment of a subset of hematologic malignancies.

The sesquiterpene lactone parthenolide induces selective apoptosis of B-chronic lymphocytic leukemia cells in vitro.

We have studied the in vitro actions of the sesquiterpene lactone parthenolide (PTL) on cells isolated from patients with chronic lymphocytic leukemia (CLL). Dye reduction viability assays showed that the median LD50 for PTL was 6.2 μM (n=78). Fifteen of these isolates were relatively resistant to the conventional agent chlorambucil but retained sensitivity to PTL. Brief exposures to PTL (1–3 h) were sufficient to induce caspase activation and commitment to cell death. Chronic lymphocytic leukemia cells were more sensitive towards PTL than were normal T lymphocytes or CD34+ haematopoietic progenitor cells. The mechanism of cell killing was via PTL-induced generation of reactive oxygen species, resulting in turn in a proapoptotic Bax conformational change, release of mitochondrial cytochrome c and caspase activation. Parthenolide also decreased nuclear levels of the antiapoptotic transcription factor nuclear factor-kappa B and diminished phosphorylation of its negative regulator IκB. Killing of CLL cells by PTL was apparently independent of p53 induction. This is the first report showing the relative selectivity of PTL towards CLL cells. The data here warrant further investigation of this class of natural product as potential therapeutic agents for CLL.

Ligation of CD8α on human natural killer cells prevents activation‐induced apoptosis and enhances cytolytic activity

It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric α/α form are more cytotoxic than their CD8– counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8– NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8α+ cells after lysis of the same targets. We report that cross‐linking of the CD8α chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK‐cell apoptosis from which CD8α+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK‐cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti‐MHC Class I antibody. Thus, in contrast to the CD8– subset, CD8α+ NK cells are capable of sequential lysis of multiple target cells.

Increased mature and immature CCR3 messenger RNA+ eosinophils in bone marrow from patients with atopic asthma compared with atopic and nonatopic control subjects

BACKGROUND Eosinophil infiltration of the bronchial mucosa is characteristic of asthma. Eosinophils differentiate from CD34(+) progenitors. Animal models suggest cooperation between IL-5 and eotaxin to allow rapid mobilization of a pool of bone marrow eosinophils followed by recruitment to the airway mucosa. OBJECTIVE The purpose of this study was to enumerate CD34(+) cell numbers in blood and bone marrow from atopic asthmatics and control subjects and to test the hypothesis that there is an increased bone marrow pool of CCR3(+) eosinophils in patients with atopic asthma, as compared with control subjects. METHODS Bone marrow aspirates and peripheral blood were obtained from volunteers with asthma and control volunteers. CD34(+) cell numbers were evaluated by flow cytometry, and eosinophil colony-forming activity was evaluated by methylcellulose cultures. Mature eosinophils, eosinophil myelocytes, metamyelocytes, and band forms (immature eosinophils) were enumerated by morphologic findings and immunocytochemistry for eosinophil cationic protein. CCR3 and eotaxin mRNA expression was examined by in situ hybridization, and protein expression was examined by immunocytochemistry. CCR3(+) cells were further identified with Chromotrope 2R staining. RESULTS CD34(+) cell numbers in bone marrow were increased in atopic subjects. Numbers of eosinophil colony-forming units in blood and bone marrow did not differ between groups. Percentages of both mature and immature eosinophils were increased in bone marrow from patients with atopic asthma, but not atopic patients with no asthma or normal control subjects. CCR3 was expressed by immature and mature bone marrow eosinophils. Eotaxin was expressed by bone marrow cells from all 3 groups, but there was no increase in subjects with asthma. CONCLUSION These findings suggest that in humans there is an increased bone marrow pool of CCR3(+) mature and immature eosinophils available for rapid mobilization in subjects with asthma but not in atopic subjects with no asthma.

IL-3, IL-5, granulocyte-macrophage colony-stimulating factor receptor α-subunit, and common β-subunit expression by peripheral leukocytes and blood dendritic cells

Abstract Background: IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors consist of cytokine-specific α-subunits, which associate with a shared signalling common β-subunit (βc) to form a high-affinity complex. The expression of IL-3, IL-5, and GM-CSF is upregulated in atopic inflammation, and these cytokines are thought to contribute to pathology through mechanisms that include eosinophil activation. Objective: We sought to examine the distribution of receptor expression between cells relevant to allergic inflammation from individual subjects and to compare atopic and nonatopic individuals. Methods: Peripheral blood was obtained from atopic and nonatopic volunteers. Cytokine-receptor expression was examined by flow cytometry with monoclonal antibodies specific for α-subunits and βc in combination with phenotypic markers for eosinophils, basophils, neutrophils, dendritic cells, monocytes, and T cells. Results: Using a ligand-independent system, we confirmed the cellular distribution of IL-5Rα, IL-3Rα, and GM-CSFRα. IL-3Rα and GM-CSFRα were detected on high-affinity IgE receptor blood dendritic cells. βc expression was detected on basophils, eosinophils, neutrophils, and, at low levels, on monocytes and dendritic cells. There was intense staining of basophils for IL-3Rα relative to IL-5Rα, GM-CSFRα, and βc, whereas eosinophil-staining intensity was similar for IL-3Rα, IL-5Rα, GM-CSFRα, and βc. There were no significant differences between atopic and nonatopic subjects in cytokine-receptor staining. Conclusion: IL-3Rα and GM-CSRα are shown on a newly defined population of FcϵRI-high dendritic cells. The intense staining of basophils for IL-3Rα, relative to that of IL-5Rα and GM-CSFRα, is in contrast to eosinophils from the same subjects and may explain the higher sensitivity of basophils to IL-3 compared with IL-5 and GM-CSF. We found no evidence for downregulation of receptor expression in atopic compared with nonatopic subjects, suggesting that these receptors remain accessible as potential targets for therapeutic intervention in atopic allergic disease. (J Allergy Clin Immunol 1998;101:677-82.)

2-phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells

We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 microM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.

Leukemic Priming of Resting NK Cells Is Killer Ig-like Receptor Independent but Requires CD15-Mediated CD2 Ligation and Natural Cytotoxicity Receptors

Resting human NK cells require a two-stage activation process that we have previously described as “priming” and “triggering.” NK-sensitive tumor cells provide both priming and triggering signals. NK-resistant tumors evade lysis, mostly by failure to prime; however, we recently reported a tumor cell line (CTV-1) that primes resting NK cells but fails to trigger lysis. In this article, we report two additional leukemia cell lines that prime NK cells but are resistant to lysis. Tumor-mediated NK priming is via CD2 binding to a ligand within CD15 on the tumor cell. NK-resistant RAJI cells became susceptible to NK lysis following transfection and expression of CD15. Blockade of CD15 on K562 cells or on CD15+ RAJI cells significantly inhibited lysis, as did blockade of CD2 on resting NK cells. NK priming via CD2 induced CD16 shedding, releasing CD3ζ to the CD2, leading to its phosphorylation and the subsequent phosphorylation of linker for activation of T cells and STAT-5 and synthesis of IFN-γ. Blockade of C-type lectin receptors significantly suppressed the tumor-mediated priming of NK cells, whereas blockade of Ig-superfamily–like receptors had no effect at the NK-priming stage. Tumor priming of resting NK cells was irrespective of HLA expression, and blockade of HLA–killer Ig-like receptor interactions did not influence the incidence or degree of priming. However, CD15–CD2 interactions were critical for NK priming and were required, even in the absence of HLA-mediated NK inhibition. Tumor-mediated priming led to a sustained primed state, and the activated NK cells retained the ability to lyse NK-resistant tumors, even after cryopreservation.