Identification of Ly2 members as antimicrobial peptides from zebrafish Danio rerio.

The emergence of multidrug-resistant (MDR) microbes caused by overuse of antibiotics leads to urgent demands for novel antibiotics exploration. Our recent data showed that Ly2.1-3 (a novel lymphocyte antigen 6 (Ly6) gene cluster) were proteins with cationic nature and rich in cysteine content, that are characteristic of antimicrobial peptides (AMPs) and their expression were all significantly up-regulated after challenge with lipopolysaccharide (LPS). These strongly suggested that Ly2.1-3 are potential AMPs, but firm evidence are lacking. Here, we clearly showed that the recombinant proteins of Ly2.1-3 were capable of killing Gram-negative bacteria Aeromonas hydrophila and Escherichia coli, while they had little bactericidal activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis We also showed that recombinant proteins Ly2.1-3 (rLy2.1-3) were able to bind to the Gram-negative bacteria A. hydrophila, E. coli and the microbial signature molecule LPS, but not to the Gram-positive bacteria S. aureus and B. subtilis as well as the microbial signature molecule LTA. Moreover, the Scatchard analysis revealed that rLy2.1-3 could specifically bind to LPS. Finally, we found that Ly2.1-3 were not cytotoxic to mammalian cells. All these together indicate that Ly2.1-3 can function as AMPs.

Isolation and characterization of nontubular sca-1+lin- multipotent stem/progenitor cells from adult mouse kidney.

Tissue engineering and cell therapy approaches aim to take advantage of the repopulating ability and plasticity of multipotent stem cells to regenerate lost or diseased tissue. Recently, stage-specific embryonic kidney progenitor tissue was used to regenerate nephrons. Through fluorescence-activated cell sorting, microarray analysis, in vitro differentiation assays, mixed lymphocyte reaction, and a model of ischemic kidney injury, this study sought to identify and characterize multipotent organ stem/progenitor cells in the adult kidney. Herein is reported the existence of nontubular cells that express stem cell antigen-1 (Sca-1). This population of small cells includes a CD45-negative fraction that lacks hematopoietic stem cell and lineage markers and resides in the renal interstitial space. In addition, these cells are enriched for beta1-integrin, are cytokeratin negative, and show minimal expression of surface markers that typically are found on bone marrow-derived mesenchymal stem cells. Global gene profiling reveals enrichment for many genes downstream of developmental signaling molecules and self-renewal pathways, such as TGF-beta/bone morphogenic protein, Wnt, or fibroblast growth factor, as well as for those that are involved in specification of mesodermal lineages (myocyte enhancer factor 2A, YY1-associated factor 2, and filamin-beta). In vitro, they are plastic adherent and slowly proliferating and result in inhibition of alloreactive CD8(+) T cells, indicative of an immune-privileged behavior. Furthermore, clonal-derived lines can be differentiated into myogenic, osteogenic, adipogenic, and neural lineages. Finally, when injected directly into the renal parenchyma, shortly after ischemic/reperfusion injury, renal Sca-1(+)Lin(-) cells, derived from ROSA26 reporter mice, adopt a tubular phenotype and potentially could contribute to kidney repair. These data define a unique phenotype for adult kidney-derived cells, which have potential as stem cells and may contribute to the regeneration of injured kidneys.

Two structurally related rat Ly49 receptors with opposing functions (Ly49 stimulatory receptor 5 and Ly49 inhibitory receptor 5) recognize nonclassical MHC class Ib-encoded target ligands.

The Ly49 family of lectin-like receptors in rodents includes both stimulatory and inhibitory members. Although NK alloreactivity in mice is regulated primarily by inhibitory Ly49 receptors, in rats activating Ly49 receptors are equally important. Previous studies have suggested that activating rat Ly49 receptors are triggered by polymorphic ligands encoded within the nonclassical class Ib region of the rat MHC, RT1-CE/N/M, while inhibitory Ly49 receptors bind to widely expressed classical class Ia molecules encoded from the RT1-A region. To further investigate rat Ly49-mediated regulation of NK alloreactivity, we report in this study the identification and characterization of two novel paired Ly49 receptors that we have termed Ly49 inhibitory receptor 5 (Ly49i5) and Ly49 stimulatory receptor 5 (Ly49s5). Using a new mAb (mAb Fly5), we showed that Ly49i5 is an inhibitory receptor that recognizes ligands encoded within the class Ib region of the u and l haplotypes, while the structurally related Ly49s5 is an activating receptor that recognizes class Ib ligands of the u haplotype. Ly49s5 is functionally expressed in the high NK-alloresponder PVG strain, but not in the low alloresponder BN strain, in which it is a pseudogene. Ly49s5 is hence not responsible for the striking anti-u NK alloresponse previously described in BN rats (haplotype n), which results from repeated alloimmunizations with u haplotype cells. The present studies support the notion of a complex regulation of rat NK alloreactivity by activating and inhibitory Ly49 members, which may be highly homologous in the extracellular region and bind similar class Ib-encoded target ligands.

Identification of SLURP-1 as an epidermal neuromodulator explains the clinical phenotype of Mal de Meleda.

Mal de Meleda is an autosomal recessive inflammatory and keratotic palmoplantar skin disorder due to mutations in the ARS B gene, encoding for SLURP-1 (secreted mammalian Ly-6/uPAR-related protein 1). SLURP-1 belongs to the Ly-6/uPAR superfamily of receptor and secreted proteins, which participate in signal transduction, immune cell activation or cellular adhesion. The high degree of structural similarity between SLURP-1 and the three fingers motif of snake neurotoxins and Lynx1 suggests that this protein interacts with the neuronal acetylcholine receptors. We found that SLURP-1 potentiates the human alpha 7 nicotinic acetylcholine receptors that are present in keratinocytes. These results identify SLURP-1 as a secreted epidermal neuromodulator which is likely to be essential for both epidermal homeostasis and inhibition of TNF-alpha release by macrophages during wound healing. This explains both the hyperproliferative as well as the inflammatory clinical phenotype of Mal de Meleda.

Ly-6I, a new member of the murine Ly-6 superfamily with a distinct pattern of expression.

A new member of the mouse Ly-6SF, designated Ly-6I, has been isolated as a gene homologous to a segment of the Ly-6C gene. A single allelic difference in the mature protein sequence was identified, which is similar to other Ly-6SF members. Ly-6I mRNA has been detected in a wide range of tissues and cell lines, and a rabbit polyclonal Ab has been used to determine that Ly-6I protein is present at a low constitutive level on cell lines from several different lineages. In contrast to Ly-6C and Ly-6A/E, the Ly-6I gene is only weakly responsive to IFNs. Expression in vivo is most abundant on bone marrow populations and is coexpressed with Ly-6C on granulocytes and macrophages. However, Ly-6I is also expressed on immature B cell populations that do not express Ly-6C. Expression on mature B cells in spleen is uniformly low. Similarly, Ly-6I is expressed on TCRlow/int, but not TCRhigh, thymocytes. Ly-6I is re-expressed on Ly-6Chigh T cells in the periphery. Thus, Ly-6I may be a useful marker to define maturation stages of both T and B lymphocytes as well as subsets of monocytes and granulocytes.

L3T4(CD4)-, Lyt-2(CD8)- and Mac-1(CD11b)-phenotypic leukocytes in murine cryptococcal meningoencephalitis.

An immunohistological study of L3T4(CD4)+ and LYT-2(CD8)+ lymphocytes, Mac-1(CD11b)+ monocytes and granulocytes in experimental murine cryptococcal meningoencephalitis was conducted. To assess the concomitant inflammatory reaction in an extracerebral site, livers were examined in parallel. Mice were infected i.v. with Cryptococcus neoformans, group A/D, and organs were examined immunohistologically for CD4-, CD8- and monocyte- and granulocyte-specific CD11b-phenotypic leukocytes over a period of 60 days. Intracerebrally, agglomerations of cryptococci formed pseudocysts that were surrounded by CD4+ and CD8+ lymphocytes at the end of the second week post-infection, followed by the invasion of monocytes and granulocytes into the lesions. After the fourth week post-infection, most of the invaded lesions were transformed into glious scars. Meningitis was usually marked and showed a homogenous distribution of CD4-, CD8- and CD11b-phenotypic cells, with a predominance of monocytes and CD4+ lymphocytes. Inflammatory infiltrates in the liver were found already 4 days post-infection. CD4+ lymphocytes and monocytes were distributed homogeneously in the infiltrates, with a lower number of CD8+ lymphocytes being located rather in the periphery of the infiltrates. Comparing leukocyte kinetics in brain and liver, an important observation was the delayed immigration of immune cells at the intracerebral cryptococcal lesions as compared with the liver, and the different migration patterns of T-lymphocyte subgroups and macrophages. These results suggest that there are differential leukocyte migration patterns in the liver and brain following disseminated cryptococcosis. The immunological aspects of the observed leukocyte kinetics are discussed.

Active tyrosine phosphatase in immunoprecipitates of multiple isoforms of Ly-5.

Using tumour cell lines expressing specific isoforms of murine Ly-5 (molecular weights of 180,000, 200,000 and 240,000) we find that all forms of Ly-5 and immuno-affinity purified forms of Ly-5 contain tyrosyl phosphatase activity. These results demonstrate that these isoforms of Ly-5 belong to the same family of functional receptor-linked tyrosine phosphatases as the human leukocyte common antigen. CD45.

Pan natural killer cell monoclonal antibodies and their relationship to the NK1.1 antigen.

The study of natural killer (NK) has been difficult because they account for a small percentage of peripheral blood and splenic lymphocytes and the paucity of NK specific antigens that have been identified. We have isolated pure populations of C57BL/6 (H-2b) NK cells using the IgG2b monoclonal antibody PK136 (anti-NK1.1). These NK1.1+ cells were used to immunize 129/J (H-2b) mice, and in this report, we describe three new NK specific monoclonal antibodies (SW3A4(IgM), SW4B12(IgG1), and SW2B4(IgG2b] and their relationship to the known murine NK antigen NK1.1. We have further characterized the NK1.1 antigen as a 39 kd molecule which is coded for by a gene which appears to map to chromosome 6.

CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells.

A novel cell surface antigen has been identified on a wide range of lymphoid cells and erythrocytes. A mAb YTH 53.1 (CD59) against this antigen enhanced the lysis of human red cells and lymphocytes by homologous complement. Studies of reactive lysis using different species of C56, and of whole serum used as a source of C7-9, indicated that the inhibitory activity of the CD59 antigen is directed towards the homologous membrane attack complex. CD59 antigen was purified from human urine and erythrocyte stroma by affinity chromatography using the mAb YTH 53.1 immobilized on Sepharose, and, following transient expression of a human T cell cDNA library in COS cells, the corresponding cDNA also identified using the antibody. It was found that the CD59 antigen is a small protein (approximately 20 kD as judged by SDS-PAGE, 11.5 kD predicted from the isolated cDNA) sometimes associated with larger components (45 and 80 kD) in urine. The sequence of CD59 antigen is unlike that of other complement components or regulatory proteins, but shows 26% identity with that of the murine LY-6 antigen. CD59 antigen was released from the surface of transfected COS cells by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor; it is therefore likely to be absent from the surface of affected erythrocytes in the disease paroxysmal nocturnal hemoglobinuria.

Tumor necrosis factor synergistically acts with IFN-gamma to regulate Ly-6A/E expression in T lymphocytes, thymocytes and bone marrow cells.

The Ly-6 alloantigens represent a family of phosphatidylinositol anchored proteins that function in the process of T lymphocyte activation and whose expression are often induced on T and B lymphocytes after activation by mitogens or Ag. Previous studies have shown that the induction of Ly-6 alloantigens in T cells is at least in part due to the action of IFN-alpha/beta or IFN-gamma. In the present report, we have demonstrated that IFN-gamma also induced Ly-6 molecules on B lymphocytes, several B cell tumors, and bone marrow cells. Furthermore, we now show that TNF also participates in the induction of at least one of the Ly-6 proteins, Ly-6A/E. TNF was found to synergize with IFN-gamma to induce Ly-6A/E expression in thymocytes, T lymphocytes, bone marrow cells, but not B lymphocytes. For T lymphocytes, the synergistic induction of Ly-6A/E by TNF was restricted to cells from the Ly-6.1 haplotype, whereas IFN-gamma was sufficient to fully induce Ly-6A/E expression in cells from the Ly-6.2 haplotype. This result is consistent with the notion that there is more complex regulation of the Ly-6A/E molecules in T cells obtained from the Ly-6.1 haplotype. For T lymphocytes from BALB/c (Ly-6.1) mice, Ly-6A/E, but not Ly-6C, molecules were synergistically induced by IFN-gamma and TNF. The induction of Ly-6A/E molecules on BALB/c T cells resulted in an enhanced capacity to activate these cells through the Ly-6 T cell activation pathway. One transformed T cell line, 5.1.2, was also identified whose Ly-6A/E molecules were synergistically induced by IFN-gamma and TNF. Optimal expression of Ly-6A/E molecules on 5.1.2 cells required continuous culture of this cell line with these two cytokines and resulted in the detection of optimal levels of cytoplasmic Ly-6A/E mRNA by Northern blot analysis. This latter result suggests that IFN-gamma and TNF regulate Ly-6A/E at the level of transcription and/or mRNA stabilization.

Remethylation at sites 5' of the murine Lyt-2 gene in association with shutdown of Lyt-2 expression.

We have used hybridomas made by fusing the Lyt-2- AKR thymoma, BW5147, to Lyt-2+ SJL/J lymph node cells to study the regulation of Lyt-2 expression. Fusions of this type yielded hybridomas, the majority of which failed to express Lyt-2. In the minority of hybridomas that did express surface Lyt-2, expression was transient and greatly diminished in terms of molecules of Lyt-2 per cell. Lack of Lyt-2 expression was not due to loss of the gene encoding this cell surface molecule; rather, negative regulation of Lyt-2 appeared to be at the level of transcription (i.e., no Lyt-2 transcripts were detected in these hybridomas). We have shown that the Lyt-2 gene is undermethylated in normal Lyt-2+ T cells, whereas the gene is heavily methylated in Lyt-2- liver cells and in BW5147. Loss of Lyt-2 expression in (BW5147 x Lyt-2+ SJL/J lymph node cell) hybridomas was associated with remethylation of DNA within the Lyt-2 gene and at sites 5' of the Lyt-2 gene.

The 33,000 protein precipitated by Ly-6A.2-specific antibodies is not associated with the Ly-6 polymorphism.

In this study, the relative mass of the Ly-6A.2 antigen was shown to be 12,000-14,000, in contrast to initial studies which showed the relative mass to be 33,000. Using polymorphic Ly-6-specific antibodies, the 33,000 molecules could be immunoprecipitated from surface-iodinated thymocytes of Ly-6A.2+, Ly-6A.2- strains and a Ly-6A.2- mutant cell line BW(Thy-1-e). This clearly demonstrated that 33,000 molecules were not associated with the Ly-6 polymorphism. By contrast, when biosynthetically labeled Ly-6A.2+ spleen cell lysates were analyzed, the major species immunoprecipitated by the polymorphic Ly-6A.2-specific antibody was 12,000-14,000, although a minor 33,000 species were also evident. The Ly-6A-specific antibody D7 which detects a monomorphic epitope on the Ly-6A molecule could immunoprecipitate the 12,000-14,000 molecules from surface-labeled cells. By contrast, the Ly-6A.2-specific antibodies detecting the polymorphic Ly-6A.2 determinant could not, though the reasons for this difference are not clear. Thus 12,000-14,000 molecules were only immunoprecipitated from Ly-6A.2+ cells, whereas 33,000 molecules were precipitated from both Ly-6A.2+ cells and Ly-6A.2- cells. These findings suggest that the 33,000 molecules immunoprecipitated by 5041-24.2 are most likely to be an unrelated protein, possibly cross-reactive with some Ly-6A.2 antibodies.

Co-immunoprecipitation of the Ly-5 molecule and an endogenous protease: a proteolytic system requiring a reducing agent and Ca2+1.

Sodium [3H]borohydride- and [35S]methionine-labeled Ly-5 molecules from mouse thymocytes and T lymphoma cells were isolated with specific antibody and Staphylococcus aureus Cowan I (SaCI) strain; after extensive washing of the complexes, elution with Laemmli's reducing buffer (0.05 M Tris [pH 6.8 or 6.0], 4% sodium dodecyl sulfate [SDS], and 2% 2-mercaptoethanol [2-ME]) resulted in partial breakdown of the isolated Ly-5 molecules from a Mr = 175,000 to 150,000. Other proteins present during the elution step showed no evidence of proteolysis. 2-ME and SDS were required for proteolysis; although addition of exogenous Ca2+ during elution was not necessary, both EDTA and EGTA inhibited breakdown of the molecule that could be overcome by excess Ca2+. Of a variety of protease inhibitors and thiol-reactive agents tested, only TAME and oxidized glutathione blocked proteolysis almost completely. SaCI, serum, and contaminating antibodies were ruled out as the source of the proteolytic activity. More stringent preclearing and washing conditions did not eliminate endogenous proteolysis of the Ly-5 molecule. The endogenous proteolytic fragment had a Mr distinct from the tryptic fragment of the Ly-5 molecule. We conclude that the Ly-5 molecule may be autolytic or tightly associated with a distinct cellular protease.

The polypeptide structure and assembly of Ly-2/3 heterodimers.

Mild reduction of mature, thymic Ly-2/3 heterodimers of Mr 67 000 resulted in dissociation into three individual polypeptide chains, alpha, alpha', and beta, of respective Mr values 38 000, 35 000, and 30 000. The alpha and alpha' chains were both immunoprecipitated by a monoclonal antibody directed to the Ly-2.1 epitope whereas the Ly-3.1 antibody bound only the beta chain. The possibility that the alpha and beta chains of each heterodimer established their interchain links within a labile precursor protein in which alpha and beta segments were fused was considered but discounted by the finding that in mice heterozygous for both Ly-2 and Ly-3 loci, the Ly-2 product of one chromosome was not exclusively joined to Ly-3 structures coded by the same chromosome. By utilizing ionic detergents which selectively alter the charge of intrinsic membrane proteins, both Ly-2 and Ly-3 polypeptides were shown to have membrane insertion sites. It is suggested that as a consequence of their likely synthesis on membrane-bound polysomes, newly synthesized Ly-2 and Ly-3 structures accumulate within the same subcellular compartment - the membranes of the rough endoplasmic reticulum. Their elevated concentration within this space may facilitate a low affinity binding interaction between Ly-2 and Ly-3 which is later stabilized by interchain disulfide bond formation.

Structure and biosynthesis of Lyt-1.

Two-dimensional chymotryptic peptide maps of Lyt-1.1 and Lyt-1.2 from Lyt-1 congenic thymocytes labeled with 125I in the usual way before lysis did not significantly differ, but there was a characteristic difference between maps of Lyt-1.1 and Lyt-1.2 obtained from 125I-labeled solubilized membrane fragments. We conclude that the Lyt-1 locus of chromosome 19 includes the protein-structural gene for Lyt-1. This conclusion is further supported by evidence with 35S-cysteine-labeled thymocytes: Thus an early 62K intermediate form, and a 60K form from tunicamycin-treated cells devoid of N-linked and O-linked carbohydrates, were precipitated by Lyt-1 alloantibody, which implies that the allo-specificity of Lyt-1 glycoprotein (67K) resides partly or wholly in protein.

Spontaneous release of the Leu-2 (T8) molecule from human T cells.

Sensitive enzyme-linked immunosorbent assays (ELISA) for the detection of human T cell antigens in soluble form have been developed. The assays use mouse monoclonal antibodies and specific anti-Leu sera prepared in rabbits by immunizing with Leu antigens absorbed to monoclonal antibody affinity columns. With these assays, Leu-1, -2, and -3 antigen signals from extracts of as few as 5 X 10(3) cells could be detected. When culture supernatants from various cell lines were tested, Leu-2 antigen, but not Leu-1 or Leu-3, was found to be present. Leu-2 antigen was present only in supernatants from T cell lines that expressed Leu-2 on their cell surface. Leu-2 antigen accumulated progressively in the supernatant of low density culture and its presence did not depend on cell proliferation or on fetal calf serum in the culture medium. The Leu-2 antigen in the supernatant was found to have only one Leu-2a determinant, whereas Leu-2 antigen from cell extracts had at least two determinants. The Leu-2 molecule was effectively purified from supernatant with an anti-Leu-2a affinity column. The purified Leu-2 antigen from supernatant of HPB-ALL cells was a single polypeptide chain of 27,000 mol wt, whereas Leu-2 antigen present on HPB-ALL cell surface was composed of two or more identical polypeptide chains of 33,000 mol wt linked by disulfide bonds. Normal human sera and sera from leukemia patients were also examined for the presence of the Leu-2 antigen. Normal human sera contained low levels of Leu-2 antigen but sera from Leu-2-positive leukemia patients had high levels. These results indicate that Leu-2 antigen is released from human T cells under physiological conditions.

Molecular weight determination of two genetically linked cell surface murine antigens: ThB and Ly-6.

Various murine tumor lines were screened by FACS analysis for the surface antigens ThB and Ly-6.2. Positive cell lines were used for immunoprecipitation studies. A monoclonal ThB-specific antibody immunoprecipitated a unique acidic protein of approximately 16 000 daltons from several positive tumors and from concanavalin A (Con-A) and LPS activated splenic lymphocytes. Monoclonal Ly-6.2-specific antibody was used to immunoprecipitate a 33 500 dalton protein that was shown to exist in four similarly sized forms with different basic charges. In the course of these studies, the apparent molecular weight of the surface antigen T 30, immunoprecipitated with a monoclonal T 30-specific antibody from the cell line EL4, was found to be approximately 25 000 daltons.