A potent immunosuppressive retroviral peptide: cytokine patterns and signaling pathways.

A synthetic bioactive peptide composed of 17 amino acids (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane envelope proteins induces not only significant immunoregulatory functions but also exhibits Th1-inhibiting properties, as described by its ability to suppress cell-mediated immunity and inhibit the production of interleukin (IL) 12, IL-2, gamma interferon, and tumor necrosis factor alpha, while enhancing IL-10. An important molecular mechanism responsible for the observed cytokine profiles by CKS-17 is provided by our findings demonstrating that this small peptide activates several intracellular signaling molecules, i.e., elevates intracellular cyclic adenosine monophosphate (cAMP) levels, and induces phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, mitogen-activated protein kinase/ERK kinase (MEK), protein kinase D, Raf1, and phospholipase C gamma1 (PLCgamma1). The activation of ERK1/2 is via the PLCgamma1-protein kinase C-Raf1-MEK signaling cascade. The activation of both ERK1/2 and cAMP appears to be via a mechanism sensitive to AG879, a receptor tyrosine kinase inhibitor, but not to AG825, AG1296, or AG1478. Furthermore, phosphoinositide-3 kinase appears to mediate the CKS-17-induced activation of ERK1/2, but not of cAMP. A specific amino acid sequence as well as the dimerization of this peptide is required to confer these biological activities. The results obtained are compelling and reproducible. This highly conserved molecule may enable us to understand a basic mechanism(s) of intracellular signaling pathways, regulation of Th1/Th2 cytokines, immunosuppression, and immunologic tolerance.

Autoimmune disease: is it a disorder of the microenvironment?

Systemic lupus erythematosus (SLE) is a common systemic autoimmune disease that involves several vital organs including the cardiovascular system, joints, and kidneys. The pathology is characterized by accumulation of autoreactive lymphocytes that attack the patients' own tissues, secretion of autoantibodies and deposition of immune complexes in vital organs. Chronic widespread inflammation is the hallmark of SLE and the target of current therapy. According to recent theories, intonating immune circuits of inflammatory cytokines and immune cells constitute highly specialized targets for SLE therapy, which nonetheless consists for the most part of anti-inflammatory medications and cytotoxic drugs. For advanced autoimmune disorders, cell therapy aiming at introducing "healthy" stem cells has been promising, keeping in mind that in its current state, stem cell therapy is reserved for the most advanced diseases refractory to traditional therapy. Ongoing studies in our laboratories examined the role of the bone marrow microenvironment, in particular, mesenchymal stem cells (MSCs) in the etiopathogenesis of SLE. Specifically, we are testing the hypothesis that, in human SLE mouse model, marrow MSCs are defective structurally and functionally. Preliminary data indicate that structural and functional defects in MSC population from an autoimmune mouse model for human SLE may contribute to this pathology and consequently present a target for cell therapy.

Immunodeficiency, radiosensitivity, and the XCIND syndrome.

Through the analysis of a rare disorder called ataxia-telangiectasia (A-T), many important biological lessons have been gleaned. Today, it is clear that the underlying defect of A-T lies in the nucleus, as an inability to repair or process double strand breaks. More important, by the A-T phenotype now allows us to appreciate a much more general distinction between immunodeficiencies that are radiosensitive and those that are not.

Replacement of recipient stromal/mesenchymal cells after bone marrow transplantation using bone fragments and cultured osteoblast-like cells.

Abstract We present our experience on treatment of three children with potentially fatal diseases using a unique protocol for non-myeloablative bone marrow transplantation. The protocol was designed to promote engraftment of bone marrow stromal/mesenchymal cells (SC/MSCs) based on the knowledge from preclinical models over the last three decades. Accordingly, our protocol is the first to test the use of bone fragments as an ideal vehicle to transplant such cells residing in the bone core. Because of the paucity of knowledge for optimum transplantation of SC/MSCs in humans, we used a multifaceted approach and implanted bone fragments both intraperitoneally and directly into bone on day 0 of BMT. We also infused cultured donor osteoblast-like cells intravenously post-BMT. We were able to achieve high levels of stroma cell engraftment as defined by molecular analyses of bone biopsy specimens.

Nonmyeloablative bone marrow transplantation of BXSB lupus mice using fully matched allogeneic donor cells from green fluorescent protein transgenic mice.

Male BXSB mice, a mouse model of systemic lupus erythematosus, were given bone marrow transplants (BMT) at 20 wk of age using MHC-matched donor cells and nonmyeloablative conditioning (550 cGy irradiation). Transplanted mice and irradiation controls were followed for a period of 20 wk. Mice transgenic for green fluorescent protein were used as donors to allow tracking of donor cells and a determination of chimerism. Radiation controls had reduced renal pathology at 10 wk posttransplant, but not at 20 wk compared with untreated mice, while nonmyeloablative BMT mice had significantly reduced pathology at both time intervals. The monocytosis characteristic of older BXSB mice was also reduced by BMT, but the treatment did not prevent production of Ab to dsDNA. A stable chimerism of 24-40% donor CD45-positive cells was achieved in spleen and bone marrow, and there was no evidence of clinical graft vs host disease. Donor cells were detected in most recipient organs, notably the thymus and renal glomeruli. The results suggest that complete depletion of mature lymphocytes or of progenitor stem cells is not required to control lupus nephritis in BXSB mice.

Positive and negative selection of T cell repertoires during differentiation in allogeneic bone marrow chimeras.

T cells acquire immune functions during expansion and differentiation in the thymus. Mature T cells respond to peptide antigens (Ag) derived from foreign proteins when these peptide Ag are presented on the self major histocompatibility complex (MHC) molecules but not on allo-MHC. This is termed self-MHC restriction. On the other hand, T cells do not induce aggressive responses to self Ag (self-tolerance). Self-MHC restriction and self-tolerance are not genetically determined but acquired a posteriori by positive and negative selection in the thymus in harmony with the functional maturation. Allogeneic bone marrow (BM) chimera systems have been a useful strategy to elucidate mechanisms underlying positive and negative selection. In this communication, the contribution of BM chimera systems to the investigation of the world of T-ology is discussed.

Negative regulation of interleukin-12 production by a rapamycin-sensitive signaling pathway: a brief communication.

Interleukin-12 (IL-12), an important cytokine in host defense against microbial pathogens, regulates natural killer and T-cell function(s) including the induction of gamma-interferon production. The major cellular sources of IL-12 are monocytes/macrophages. Bacteria, bacterial products, and intracellular parasites are the most efficient inducers of IL-12 production. In the present study we show that a signal transduction pathway sensitive to rapamycin may have an important role in the regulation/suppression of Staphylococcus aureus-induced IL-12 production in vitro. Human peripheral blood mononuclear cells, monocytes, or a human monocytic cell line THP-1 were stimulated with S. aureus Cowan strain 1 (SAC) in the presence or absence of rapamycin and investigated for production of IL-12 protein by enzyme-linked immunosorbent assay and IL-12 p40 mRNA accumulation by RNase protection assay or real-time quantitative polymerase chain reaction. The results show that rapamycin significantly enhances SAC-induced IL-12 p70 protein production and IL-12 p40 mRNA accumulation. Further the results demonstrate that wortmannin enhances SAC-induced IL-12 p40 mRNA accumulation, whereas Ly294002 does not. These data indicate that a rapamycin-sensitive signaling pathway may act as a negative feedback cascade in the regulatory mechanisms of IL-12 production.

A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

Impaired degradation of inhibitory subunit of NF-kappa B (I kappa B) and beta-catenin as a result of targeted disruption of the beta-TrCP1 gene.

beta-TrCP1 (also known as Fbw1a or FWD1) is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Although biochemical studies have suggested that beta-TrCP1 targets inhibitory subunit of NF-kappa B(I kappa B) proteins and beta-catenin for ubiquitylation, the physiological role of beta-TrCP1 in mammals has remained unclear. We have now generated mice deficient in beta-TrCP1 and shown that the degradation of I kappa B alpha and I kappa B beta is reproducibly, but not completely, impaired in the cells of these animals. The nuclear translocation and DNA-binding activity of NF-kappa B as well as the ability of this transcription factor to activate a luciferase reporter gene were also inhibited in beta-TrCP1-/- cells compared with those apparent in wild-type cells. The subcellular localization of beta-catenin was altered markedly in beta-TrCP1-/- cells. Furthermore, the rate of proliferation was reduced and both cell size and the percentage of polyploid cells were increased in embryonic fibroblasts derived from beta-TrCP1-/- mice compared with the corresponding wild-type cells. These results suggest that beta-TrCP1 contributes to, but is not absolutely required for, the degradation of I kappa B and beta-catenin and the consequent regulation of the NF-kappa B and Wnt signaling pathways, respectively. In addition, they implicate beta-TrCP1 in the maintenance of ploidy during cell-cycle progression.

Pyogenic bacterial infections in humans with IRAK-4 deficiency.

Members of the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) superfamily share an intracytoplasmic Toll-IL-1 receptor (TIR) domain, which mediates recruitment of the interleukin-1 receptor-associated kinase (IRAK) complex via TIR-containing adapter molecules. We describe three unrelated children with inherited IRAK-4 deficiency. Their blood and fibroblast cells did not activate nuclear factor kappaB and mitogen-activated protein kinase (MAPK) and failed to induce downstream cytokines in response to any of the known ligands of TIR-bearing receptors. The otherwise healthy children developed infections caused by pyogenic bacteria. These findings suggest that, in humans, the TIR-IRAK signaling pathway is crucial for protective immunity against specific bacteria but is redundant against most other microorganisms.

Mixed bone marrow or mixed stem cell transplantation for prevention or treatment of lupus-like diseases in mice.

Scientific analyses fortified by interpretations of immunodeficiency diseases as 'experiments of nature' have revealed the specific immune systems to be comprised of T cells subserving cell-mediated immunities plus B cells and plasma cells which produce and secrete antibodies. These two separate cellular systems regularly interact with each other to produce a coordinated defense which permits mammals to live within a sea of microorganisms that threaten the integrity and the survival of individuals. We have shown that bone marrow transplantation (BMT) can be used as a form of cellular engineering to construct or reconstruct the immune systems and cure otherwise fatal severe combined immunodeficiency. When severe aplastic anemia complicated the first BMT which was performed to cure a fatal severe combined immunodeficiency, a second BMT cured for the first time a complicating severe aplastic anemia. Subsequently, BMT has been used effectively to treat some 75 otherwise fatal diseases such as resistant leukemias, lymphomas, inborn errors of metabolism, and genetic anomalies of the hematopoietic development such as sickle cell anemia, thalassemia, congenital neutropenias, and many other diseases. More recently, we have employed BMT in mice both to cure and cause autoimmunities, and, together, these experiments showed that autoimmunities actually reside in the hematopoietic stem cells. We have also found that mixed BMT or mixed hematopoietic stem cell transplantation (HSCT) can be used to prevent and cure the most complex autoimmunities such as those occurring in BXSB mice and in (NZW x BXSB)F1 W/BF1 mice. Untreated, the former develop fulminating lethal glomerulonephritis plus numerous humoral autoimmunities. Mice of the (W/B)F1 strain develop autoimmune thrombocytopenic purpura, coronary vascular disease with myocardial infarction, glomerulonephritis, and numerous autoantibodies. All of these abnormalities are prevented or cured by mixed syngeneic (autoimmune) plus allogeneic (normal healthy) BMT or mixed peripheral blood HSCT. Thus, the most complex autoimmune diseases can be prevented or cured in experimental animals by mixed syngeneic plus allogeneic BMT or HSCT which produce stable mixed chimerism as a form of cellular engineering.

Cellular immunology in a historical perspective.

Bruton's XLA and DiGeorge syndrome patients show that two basic immune systems are distinct from each other in humans - thymus-dependent cell-mediated immunodeficiencies vs. antibody-based immunodeficiencies. The appendix-sacculus lymphoid organ of rabbits, like the bursa of Fabricius, represents a central lymphoid organ. Chronic granulomatous disease of childhood (CGD) revealed that phagocytosis killing of catalase-positive microorganisms employ oxidative burst. Bone marrow transplantation (BMT) proved life saving in severe combined immunodeficiency (SCID). The first BMT cured XSCID and the second BMT cured a complicating aplastic anemia launching BMT as a treatment of many diseases. Now 75 fatal diseases have been cured by myeloablative BMT. BMT also cured experimental autoimmune diseases. BMT alone did not cure lupus with polyarthritis in MRL/lpr mice or polyarthritis in NZB/KN mice, but BMT plus bone (stromal cell) transplants cured these diseases. Autoimmune diseases and lethal glomerulonephritis were prevented or cured in BXSB mice by mixed allogeneic plus syngeneic BMT. X-linked Hyper IgM syndrome (XHIM) was also cured by BMT from a 2-year-old MHC-matched sibling donor. Nonmyeloablative BMT plus mesenchymal stem cells (stromal cells) was effective treatment for a form of collagen-vascular disease and also a lethal form of hypophosphatasia. Mannan-binding lectin, an opsonin that activates the complement system when mutated and at low levels in blood, opens a door to frequent infections throughout childhood and adult life. This new immunodeficiency is based on genetic mutations that involve a native defense system.

Mixed peripheral blood stem cell transplantation for autoimmune disease in BXSB/MpJ mice.

We examined whether mixed allogeneic transplantation with syngeneic plus allogeneic peripheral blood stem cells (PBSCs) is sufficient to interrupt autoimmune processes in BXSB mice and confer a potential therapeutic option for the treatment of patients with autoimmune diseases. Eight-week-old BXSB mice were lethally irradiated and reconstituted with BALB/c (H-2(d))+BXSB (H-2(b)) PBSCs, in which the number of injected allogeneic progenitor cells was 5 times that of syngeneic progenitor cells. The survival of mixed PBSC chimeras (BALB/c+BXSB-->BXSB) was 80% at the age of 48 weeks, whereas that of full chimeras (BALB/c-->BXSB) was 90%. Mixed PBSC transplantation (PBSCT) prevented the production of anti-DNA antibodies and the development of lupus nephritis in BXSB recipients and induced tolerance to both allogeneic and syngeneic antigens. Moreover, mixed chimeras exhibited immunological functions superior to fully allogeneic chimeras. On the other hand, increases in the number of BXSB PBSCs resulted in the transfer of lupus nephritis in BXSB+BALB/c-->BALB/c mice. Thus, the number of hematopoietic progenitor cells from normal mice proved critical to the prevention of autoimmune diseases. We propose that mixed allogeneic PBSCT for the interruption of the autoimmune process can be carried out by injecting increased numbers of allogeneic normal hematopoietic progenitor cells to prevent the relapse of autoimmune diseases, although it is necessary to decide upon a minimum dose of syngeneic PBSCs to achieve the desired beneficial effects on autoimmunity.

Uncoupling protein 2 plays an important role in nitric oxide production of lipopolysaccharide-stimulated macrophages.

The expression of uncoupling protein 2 (UCP2) was reduced in macrophages after stimulation with lipopolysaccharide (LPS). The physiological consequence and the regulatory mechanisms of the UCP2 down-regulation by LPS were investigated in a macrophage cell line, RAW264 cells. UCP2 overexpression in RAW264 cells transfected with eukaryotic expression vector containing ucp2 cDNA markedly reduced the production of intracellular reactive oxygen species. Furthermore, in the UCP2 transfectant, nitric oxide (NO) synthesis, inducible NO synthase (NOS II) protein, NOS II mRNA, and NOS II promoter activity were definitely decreased after LPS stimulation compared with those in parental RAW264 or RAW264 cells transfected with the vector alone. Reporter assays suggested that an enhancer element was located in the region of intron 2 of the UCP2 gene and that the UCP2 expression was down-regulated not by the 7.3-kb promoter region but by the 5' region of the UCP2 gene containing two introns. Deletion of intron 2 resulted in the low transcriptional activities and abolishment of the LPS-associated negative regulation. In addition, the mRNA expression of transfected UCP2 was suppressed in RAW264 cells transfected with expression vector containing UCP2 genomic DNA, but was markedly increased in cells transfected with the vector containing UCP2 intronless cDNA. These findings suggest that the LPS-stimulated signals suppress UCP2 expression by interrupting the function of intronic enhancer, leading to an up-regulation of intracellular reactive oxygen species, which activate the signal transduction cascade of NOS II expression, probably to ensure rapid and sufficient cellular responses to a microbial attack.

Roles of NKT cells in resistance against infection with Toxoplasma gondii and in expression of heat shock protein 65 in the host macrophages.

We investigated the roles of gamma delta T, NK, and NK1.1(+) T-like (NKT) cells in protective immunity against infection with Toxoplasma gondii. gamma delta T cells, NKT and NK cells, and NK cells in BALB/c mice were depleted by treatment with anti-TCR-gamma delta monoclonal antibody (mAb), anti-interleukin-2 receptor beta chain (IL-2R beta) mAb, and anti-asialoGM1 Ab, respectively, and these mice were infected with T. gondii. Treatment of mice with anti-TCR-gamma delta mAb aggravated toxoplasmosis, while treatment with anti-asialoGM1 Ab had no effects. Treatment with anti-IL-2R beta mAb enhanced the expression of heat shock protein 65 (HSP65) and gamma interferon (IFN-gamma) mRNA, while it inhibited interleukin-4 (IL-4) mRNA expression, ameliorating toxoplasmosis. In addition to NK cells, anti-IL-2R beta mAb eliminated cells expressing IL-2R beta and intermediate levels of CD3 (IL-2R beta(+) CD3(int)). Mice treated with anti-IL-2R beta mAb decreased the number of DX5(+) CD3(int) cells, which are considered to be equivalent to NK1.1(+)T cells in NK1.1 allele-negative strains. IL-2R beta(+) CD3(int) cells isolated from splenic and hepatic lymphoid cells were confirmed to express the TCR-V alpha 14 transcript. The magnitude of HSP65 induction in macrophages correlated with the protective potential against T. gondii infection after treatment with the antibodies, supporting our previous finding that gamma delta T cells play an essential role in the induction of HSP65 in host macrophages. Interestingly, NKT cells suppressed the expression of gamma delta T cell-induced HSP65 and IFN-gamma. Furthermore, depletion of IL-2R beta(+) CD3(int) cells suppressed the IL-4 mRNA expression. These results suggest that NKT cells may be the cells responsible for suppression of protective immunity against T. gondii infection by interfering with the gamma delta T cell-induced HSP65 expression, possibly through the generation of IL-4.