Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, rapidly fatal, autosomal recessive immune disorder characterized by uncontrolled activation of T cells and macrophages and overproduction of inflammatory cytokines. Linkage analyses indicate that FHL is genetically heterogeneous and linked to 9q21.3-22, 10q21-22, or another as yet undefined locus. Sequencing of the coding regions of the perforin gene of eight unrelated 10q21-22-linked FHL patients revealed homozygous nonsense mutations in four patients and missense mutations in the other four patients. Cultured lymphocytes from patients had defective cytotoxic activity, and immunostaining revealed little or no perforin in the granules. Thus, defects in perforin are responsible for 10q21-22-linked FHL. Perforin-based effector systems are, therefore, involved not only in the lysis of abnormal cells but also in the down-regulation of cellular immune activation.

Natural killer (NK) cells are critical in the immune response to tumor cells, virally infected cells, and bone marrow allografts. 2B4 (CD244) is expressed on all NK cells and the ligand for 2B4, CD48, is expressed on hematopoietic cells. Cross-linking 2B4 on NK cells with anti-2B4 monoclonal antibody leads to NK cell activation in vitro. Therefore, 2B4 is considered to be an activating receptor. Surprisingly, we have found, using antibody-blocking and 2B4-deficient NK cells, that NK lysis of CD48+ tumor and allogeneic targets is inhibited by 2B4 ligation. Interferon γ production by NK cells is also inhibited. Using a peritoneal tumor clearance assay, it was found that 2B4−/− mice have increased clearance of CD48+ tumor cells in vivo. Retroviral transduction of 2B4 was sufficient to restore inhibition in 2B4−/− primary NK cells. It was found that although mature NK cells express SH2D1A, in vitro–derived NK cells do not. However, both populations are inhibited by 2B4 ligation. This indicates that 2B4 inhibitory signaling occurs regardless of the presence of SH2D1A. These findings reveal a novel role for 2B4 as a non–major histocompatibility complex binding negative regulator of NK cells.

The role of IL-6 was investigated in murine ischemic acute renal failure. The renal pedicles were clamped for 17 min, and the mice were studied at various times after reperfusion. We found that serum IL-6 increased after murine ischemic renal injury. This increase was associated with increased IL-6 mRNA in the ischemic kidney but not in the contralateral kidney or the liver. Maximal IL-6 production occurred at 4 to 8 h and decreased to baseline by 24 h. Reperfusion of the kidney was required for IL-6 production. In situ hybridization and immunohistochemistry showed that macrophages infiltrated areas adjacent to the vascular bundles in the outer medulla within hours of reperfusion and showed that these macrophages produced IL-6 mRNA. For understanding how macrophages were stimulated to produce IL-6, an in vitro model in which S3 proximal tubular cells were injured by reactive oxygen species was set up. These injured cells released molecules that activated macrophages to produce IL-6 in vitro. IL-6 that was produced in response to renal ischemia was maladaptive because transgenic knockout of IL-6 ameliorated renal injury as measured by serum creatinine and histology. IL-6 transgenic knockout mice were lethally irradiated, and their bone marrow was reconstituted with wild-type IL-6 cells. Such bone marrow transfers abolished the protective effects of transgenic IL-6 knockout. It is concluded that macrophages infiltrate the area of the vascular bundles of the outer medulla, these macrophages produce IL-6, and this IL-6 exacerbates ischemic murine acute renal failure.

Summary Proper functioning of the musculo-skeletal system requires the precise integration of bones, muscles and tendons. Complex morphogenetic events ensure that these elements are linked together in the appropriate 3D configuration. It has been difficult, however, to tease apart the mechanisms that regulate tissue morphogenesis. We find that deletion of Tbx5 in forelimb (or Tbx4 in hindlimbs) specifically affects muscle and tendon patterning without disrupting skeletal development thus suggesting that distinct cues regulate these processes. We identify muscle connective tissue as the site of action of these transcription factors and show that N-Cadherin and β-Catenin are key downstream effectors acting in muscle connective tissue regulating soft-tissue morphogenesis. In humans, TBX5 mutations lead to Holt-Oram syndrome, which is characterised by forelimb musculo-skeletal defects. Our results suggest that a focus on connective tissue is required to understand the aetiology of diseases affecting soft tissue formation.

Summ2ryLarge granular lymphocyte (LGL) 1 is a cell surface glycoprotein expressed on a subset (50%) of C57BL/6 natural killer (NK) cells. Immunoprecipitation experiments reveal that the LGL-1 protein exists as a disulfide-linked 40-kD homodimer. Functional studies of LGL-1 + cells indicate that selected H-2 d target cells are not lysed efficiently by these interleukin (IL)-2-cultured NK cells. These findings suggested that LGL-1 may be a member of the Ly-49 gene family. Here we report the molecular cloning of the LGL-1 cDNA from a severe combined immunodeficient-adherent lymphokine-activated killer cell library transfected into Cos-7 cells and find LGL-1 to be homologous to the Ly-49 gene at both the nucleotide (85%) and amino acid levels (73%). Sequencing of our LGL-1 cDNA has revealed it to be nearly identical to the Ly-49G2 cDNA recently isolated by cross-hybridization with an Ly-49 probe.LGL-1 represents a type II transmembrane protein of 267 amino acids with its carboxyl end exposed extracellularly. The LGL-1 protein contains 11 highly conserved cysteine residues and a 25-amino acid transmembrane region. Southern blot analysis demonstrates that there are a number of homologous genes in mouse DNA that hybridize strongly to LGL-1. Northern analyses using poly A + RNA from LGL-1 + NK cells indicate that LGL-1 is expressed as a 1.4 kb mRNA. Two-color flow cytometry analysis (FCA) of C57BL/6 splenic NK cells demonstrates that LGL-1 and Ly-49 label overlapping subsets of cells. FCA identifies four subsets of NK cells as defined by LGL-1 versus Ly-49 staining. We have sorted these individual subsets, expanded them in IL-2, and performed cytotoxicity experiments to determine their target cell profiles in relation to class I expression. Results of these studies are complex, but indicate that Ly-49 may not be the only molecule that recognizes class I as an inhibitory signal for cytotoxicity. LGL-1 + cells also fail to lyse several H-2d-expressing tumor targets and concanavalin A lymphoblasts from BALB/c but not C57BL/6 mice. This inhibition of lysis by LGL-1 + NK cells is negated by addition of monoclonal antibody (mAb) 4Dll that recognizes the LGL-1 protein. When mAbs to the class I molecules H-2D a and H-2L a (oltot~ domains only) are added to cytotoxicity assays, LGL-1 + cells lyse H-2 d targets very effectively. Therefore, LGL-1 recognizes regions of the class I-specific molecules H-2D a and H-2L a. This specificity distinguishes LGL-1 from Ly-49, whose killing was only reversed by antibodies to H-2D d. The differential specificities recognized by LGL-1 versus Ly-49 support the hypothesis that this family of genes recognizes diverse class I molecules and regulates the lytic activity of NK cells.

Rejection of bone marrow transplants in lethally irradiated mice differs from rejection of solid tissue grafts in several respects. The genetic laws that govern rejection of solid tissue grafts often fail with hemopoietic grafts. For example, F1 hybrids between two H-2 disparate strains of mice often reject parental bone marrow cells (BMC), and conversely, marrow cells of F1 hybrids (H-2 heterozygous) are usually not rejected by either parent or an unrelated allogeneic recipient. Thus, unlike the classical MHC antigens, the hemopoietic histocompatibility (Hh) antigens relevant in marrow graft rejection are inherited in a recessive pattern. The major Hh (Hh-1) locus maps within the mouse H-2 complex between the H-2S and H-2D regions, and it can therefore be dissociated from the class-I MHC genes. Nevertheless, it is possible that class-I MHC antigens play a role in the formation or expression of Hh-1 antigens. Three models that explain the possible relationship between class-I MHC and Hh-1 genes and the noncodominant pattern of inheritance of Hh antigens are presented. The effector cells responsible for resisting BMC grafts are different from those responsible for rejection of solid tissue grafts. Three cell types, natural killer cells (CD3-, NK1.1+), cytotoxic T cells (CD3+, CD8+), and T cells with natural killer cell markers (CD3+, NK1.1+) have been implicated in the rejection of BMC grafts. Involvement of these cell types is reviewed and the relative roles played by each are discussed. Evidence supporting the existence of Hh-1 specific subsets of NK cells is presented.

We have developed a stroma-free culture system in which mouse marrow or thymus cells, known to be enriched for lymphoid progenitors, can be driven to generate natural killer (NK) cells. Culture of lineage marker (Lin)−, c-kit+, Sca2+, interleukin (IL)-2/15Rβ (CD122)− marrow cells in IL-6, IL-7, stem cell factor (SCF), and flt3 ligand (flt3-L) for 5–6 d followed by IL-15 alone for an additional 4–5 d expanded the starting population 30–40-fold and gave rise to a virtually pure population of NK1.1+, CD3− cells. Preculture in IL-6, IL-7, SCF, and flt3-L was necessary for inducing IL-15 responsiveness in the progenitors because the cells failed to significantly expand when cultured in IL-15 alone from the outset. Although culture of the sorted progenitors in IL-6, IL-7, SCF, and flt3-L for the entire 9–11-d culture period caused significant expansion, no lytic NK1.1+ cells were generated if IL-15 was not added, demonstrating a critical role for IL-15 in NK differentiation. Thus, two distinct populations of NK progenitors, IL-15 unresponsive and IL-15 responsive, have been defined. Similar results were obtained with Lin−, CD44+, CD25−, c-kit+ lymphoid progenitors obtained from adult thymus. The NK cells generated by this protocol lysed the NK-sensitive target YAC-1 and expressed markers of mature NK cells with the notable absence of Ly-49 major histocompatibility complex (MHC) receptors. However, despite the apparent lack of these inhibitory MHC receptors, the NK cells generated could distinguish MHC class I+ from class I− syngeneic targets, suggesting the existence of novel class I receptors.

The receptor 2B4 belongs to the Ig superfamily and is found on the surface of all murine natural killer (NK) cells as well as T cells displaying non-MHC-restricted cytotoxicity. Previous studies have suggested that 2B4 is an activating molecule because cross-linking of this receptor results in increased cytotoxicity and ␥-interferon secretion as well as granule exocytosis. However, it was recently shown that the gene for 2B4 encodes two different products that arise by alternative splicing. These gene products differ solely in their cytoplasmic domains. One form has a cytoplasmic tail of 150 amino acids (2B4L) and the other has a tail of 93 amino acids (2B4S). To determine the function of each receptor, cDNAs for 2B4S and 2B4L were transfected into the rat NK cell line RNK-16. Interestingly, the two forms of 2B4 had opposing functions. 2B4S was able to mediate redirected lysis of P815 tumor targets, suggesting that this form represents an activating receptor. However, 2B4L expression led to an inhibition of redirected lysis of P815 targets when the mAb 3.2.3 (specific for rat NKRP1) was used. In addition, 2B4L constitutively inhibits lysis of YAC-1 tumor targets. 2B4L is a tyrosine phosphoprotein, and removal of domains containing these residues abrogates its inhibitory function. Like other inhibitory receptors, 2B4L associates with the tyrosine phosphatase SHP-2. Thus, 2B4L is an inhibitory receptor belonging to the Ig superfamily.