Reducing sarcolipin expression improves muscle metabolism in mdx mice.

Duchenne muscular dystrophy (DMD) is an inherited muscle wasting disease. Metabolic impairments and oxidative stress are major secondary mechanisms that severely worsen muscle function in DMD. Here, we sought to determine whether germline reduction or ablation of sarcolipin (SLN), an inhibitor of sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA), improves muscle metabolism and ameliorates muscle pathology in the mdx mouse model of DMD. Glucose and insulin tolerance tests show that glucose clearance rate and insulin sensitivity were improved in the SLN haploinsufficient mdx (mdx:sln+/-) and SLN-deficient mdx (mdx:sln-/-) mice. The histopathological analysis shows that fibrosis and necrosis were significantly reduced in muscles of mdx:sln+/- and mdx:sln-/- mice. SR Ca2+ uptake, mitochondrial complex protein levels, complex activities, mitochondrial Ca2+ uptake and release, and mitochondrial metabolism were significantly improved, and lipid peroxidation and protein carbonylation were reduced in the muscles of mdx:sln+/- and mdx:sln-/- mice. These data demonstrate that reduction or ablation of SLN expression can improve muscle metabolism, reduce oxidative stress, decrease muscle pathology, and protects the mdx mice from glucose intolerance.

A functional variomics tool for discovering drug-resistance genes and drug targets.

Comprehensive discovery of genetic mechanisms of drug resistance and identification of in vivo drug targets represent significant challenges. Here we present a functional variomics technology in the model organism Saccharomyces cerevisiae. This tool analyzes numerous genetic variants and effectively tackles both problems simultaneously. Using this tool, we discovered almost all genes that, due to mutations or modest overexpression, confer resistance to rapamycin, cycloheximide, and amphotericin B. Most significant among the resistance genes were drug targets, including multiple targets of a given drug. With amphotericin B, we discovered the highly conserved membrane protein Pmp3 as a potent resistance factor and a possible target. Widespread application of this tool should allow rapid identification of conserved resistance mechanisms and targets of many more compounds. New genes and alleles that confer resistance to other stresses can also be discovered. Similar tools in other systems, such as human cell lines, will also be useful.

Effect of transfection with PLP2 antisense oligonucleotides on gene expression of cadmium-treated MDA-MB231 breast cancer cells.

Emerging evidence indicates that cadmium (Cd) is able to regulate gene expression, drastically affecting the pattern of transcriptional activity in human normal and pathological cells. We have already shown that exposure of MDA-MB231 breast cancer cells to 5 µM CdCl(2) for 96 h, apart from significantly affecting mitochondrial metabolism, induces modifications of the expression level of genes coding for members of stress response-, mitochondrial respiration-, MAP kinase-, NF-κB-, and apoptosis-related pathways. In the present study, we have expanded the knowledge on the biological effects of Cd-breast cancer cell interactions, indicating PLP2 (proteolipid protein-2) as a novel member of the list of Cd-upregulated genes by MDA-MB231 cancer cells and, through the application of transfection techniques with specific antisense oligonucleotides, we have demonstrated that such over-expression may be an upstream event to some of the changes of gene expression levels already observed in Cd-treated cells, thus unveiling new possible molecular relationship between PLP2 and genes linked to the stress and apoptotic responses.

Pathogenic Old World arenaviruses inhibit TLR2/Mal-dependent proinflammatory cytokines in vitro.

Lymphocytic choriomeningitis virus (LCMV), the prototype arenavirus, and Lassa virus (LASV), the causative agent of Lassa fever (LF), have extensive strain diversity and significant variations in pathogenicity for humans and experimental animals. The WE strain of LCMV (LCMV-WE), but not the Armstrong (Arm) strain, induces a fatal LF-like disease in rhesus macaques. We also demonstrated that LASV infection of human macrophages and endothelial cells resulted in reduced levels of proinflammatory cytokines. Here we have shown that cells infected with LASV or with LCMV-WE suppressed Toll-like receptor 2 (TLR2)-dependent proinflammatory cytokine responses. The persisting isolate LCMV clone 13 (CL13) also failed to stimulate interleukin-6 (IL-6) in macrophages. In contrast, nonpathogenic Mopeia virus, which is a genetic relative of LASV and LCMV-Arm induced robust responses that were TLR2/Mal dependent, required virus replication, and were enhanced by CD14. Superinfection experiments demonstrated that the WE strain of LCMV inhibited the Arm-mediated IL-8 response during the early stage of infection. In cells transfected with the NF-κB-luciferase reporter, infection with LCMV-Arm resulted in the induction of NF-κB, but cells infected with LCMV-WE and CL13 did not. These results suggest that pathogenic arenaviruses suppress NF-κB-mediated proinflammatory cytokine responses in infected cells.

MAL facilitates the incorporation of exocytic uroplakin-delivering vesicles into the apical membrane of urothelial umbrella cells.

The apical surface of mammalian bladder urothelium is covered by large (500-1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function and uropathogenic bacterial binding. How the uroplakin proteins are delivered to the luminal surface is unknown. We show here that myelin-and-lymphocyte protein (MAL), a 17-kDa tetraspan protein suggested to be important for the apical sorting of membrane proteins, is coexpressed with uroplakins in differentiated urothelial cell layers. MAL depletion in Madin-Darby canine kidney cells did not affect, however, the apical sorting of uroplakins, but it decreased the rate by which uroplakins were inserted into the apical surface. Moreover, MAL knockout in vivo led to the accumulation of fusiform vesicles in mouse urothelial superficial umbrella cells, whereas MAL transgenic overexpression in vivo led to enhanced exocytosis and compensatory endocytosis, resulting in the accumulation of the uroplakin-degrading multivesicular bodies. Finally, although MAL and uroplakins cofloat in detergent-resistant raft fractions, they are associated with distinct plaque and hinge membrane subdomains, respectively. These data suggest a model in which 1) MAL does not play a role in the apical sorting of uroplakins; 2) the propensity of uroplakins to polymerize forming 16-nm particles and later large 2D crystals that behave as detergent-resistant (giant) rafts may drive their apical targeting; 3) the exclusion of MAL from the expanding 2D crystals of uroplakins explains the selective association of MAL with the hinge areas in the uroplakin-delivering fusiform vesicles, as well as at the apical surface; and 4) the hinge-associated MAL may play a role in facilitating the incorporation of the exocytic uroplakin vesicles into the corresponding hinge areas of the urothelial apical surface.

Inactivation of the MAL gene in breast cancer is a common event that predicts benefit from adjuvant chemotherapy.

Dysregulation of MAL (myelin and lymphocyte protein) has been implicated in several malignancies including esophageal, ovarian, and cervical cancers. The MAL protein functions in apical transport in polarized epithelial cells; therefore, its disruption may lead to loss of organized polarity characteristic of most solid malignancies. Bisulfite sequencing of the MAL promoter CpG island revealed hypermethylation in breast cancer cell lines and 69% of primary tumors analyzed compared with normal breast epithelial cells. Differential methylation between normal and cancer DNA was confined to the proximal promoter region. In a subset of breast cancer cell lines including T47D and MCF7 cells, promoter methylation correlated with transcriptional silencing that was reversible with the methylation inhibitor 5-aza-2'-deoxycytidine. In addition, expression of MAL reduced motility and resulted in a redistribution of lipid raft components in MCF10A cells. MAL protein expression measured by immunohistochemistry revealed no significant correlation with clinicopathologic features. However, in patients who did not receive adjuvant chemotherapy, reduced MAL expression was a significant predictive factor for disease-free survival. These data implicate MAL as a commonly altered gene in breast cancer with implications for response to chemotherapy.

ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance.

The Toll-interleukin 1 receptor (TIR) superfamily, defined by the presence of an intracellular TIR domain, initiates innate immunity through activation of the transcription factor NF-kappa B, leading to the production of proinflammatory cytokines. ST2 is a member of the TIR family that does not activate NF-kappa B and has been suggested as an important effector molecule of T helper type 2 (T(H)2) responses. We show here that the membrane-bound form of ST2 negatively regulated type I interleukin 1 receptor (IL-1RI) and Toll-like receptor 4 (TLR4) but not TLR3 signaling by sequestrating the adaptors MyD88 and Mal. In contrast to wild-type mice, ST2-deficient mice failed to develop endotoxin tolerance. Thus, these results provide a molecular explanation for the function of ST2 in T(H)2 responses, as inhibition of TLRs promotes a T(H)2 response, and also identify ST2 as a key regulator of endotoxin tolerance.

Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro.

Surfactant protein A (SP-A) is the most abundant of the surfactant-associated proteins. SP-A is involved in the formation of tubular myelin, the modulation of the surface tension-reducing properties of surfactant phospholipids, the metabolism of surfactant phospholipids, and local pulmonary host defense. We hypothesized that elimination of SP-A would alter the regulation of SP-B gene expression and the formation of tubular myelin. Midtrimester human fetal lung explants were cultured for 3-5 days in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to SP-A mRNA. After 3 days in culture, SP-A mRNA was undetectable in antisense ON-treated explants. After 5 days in culture, levels of SP-A protein were also decreased by antisense treatment. SP-B mRNA levels were not affected by the antisense SP-A ON treatment. However, there was decreased tubular myelin formation in the antisense SP-A ON-treated tissue. We conclude that selective elimination of SP-A mRNA and protein results in a decrease in tubular myelin formation in human fetal lung without affecting SP-B mRNA. We speculate that SP-A is critical to the formation of tubular myelin during human lung development and that the regulation of SP-B gene expression is independent of SP-A gene expression.

Resistance of different surfactant preparations to inactivation by meconium.

A disease similar to acute respiratory distress syndrome may occur in neonates after aspiration of meconium. The aim of the study was to compare the inhibitory effects of human meconium on the following surfactant preparations suspended at a concentration of 2.5 mg/mL: Curosurf, Alveofact, Survanta, Exosurf, Pumactant, rabbit natural surfactant from bronchoalveolar lavage, and two synthetic surfactants based on recombinant surfactant protein-C (Venticute) or a leucine/lysine polypeptide. Minimum surface tension, determined with a pulsating bubble surfactometer, was increased >10 mN/m at meconium concentrations >or=0.04 mg/mL for Curosurf, Alveofact, or Survanta, >or=0.32 mg/mL for recombinant surfactant protein-C, >or=1.25 mg/mL for leucine/lysine polypeptide, and >or=20 mg/mL for rabbit natural surfactant. The protein-free synthetic surfactants Exosurf and Pumactant did not reach minimum surface tension <10 mN/m even in the absence of meconium. We conclude that surfactant activity is inhibited by meconium in a dose-dependent manner. Recombinant surfactant protein-C and leucine/lysine polypeptide surfactant were more resistant to inhibition than the modified natural surfactants Curosurf, Alveofact, or Survanta but less resistant than natural lavage surfactant containing surfactant protein-A. We speculate that recombinant hydrophobic surfactant proteins or synthetic analogs of these proteins can be used for the design of new surfactant preparations that are relatively resistant to inactivation and therefore suitable for treatment of acute respiratory distress syndrome.

Surfactant protein A (SP-A): the alveolus and beyond.

Surfactant protein A (SP-A) is the major protein component of pulmonary surfactant, a material secreted by the alveolar type II cell that reduces surface tension at the alveolar air-liquid interface. The function of SP-A in the alveolus is to facilitate the surface tension-lowering properties of surfactant phospholipids, regulate surfactant phospholipid synthesis, secretion, and recycling, and counteract the inhibitory effects of plasma proteins released during lung injury on surfactant function. It has also been shown that SP-A modulates host response to microbes and particulates at the level of the alveolus. More recently, several investigators have reported that pulmonary surfactant phospholipids and SP-A are present in nonalveolar pulmonary sites as well as in other organs of the body. We describe the structure and possible functions of alveolar SP-A as well as the sites of extra-alveolar SP-A expression and the possible functions of SP-A in these sites.

Surfactant protein SP-B counteracts inhibition of pulmonary surfactant by serum proteins.

In addition to the primary surfactant deficiency in newborns with respiratory distress syndrome (RDS), in the later course of RDS substantial protein leakage into the alveolar spaces can occur by damage to the alveolocapillary membrane. Acute lung injury results in surfactant dysfunction due in part to inhibition by serum proteins. The aim of this study was to investigate the influence of SP-B on the inhibitory effects of albumin (alb) and fibrinogen (fib) on the surface activity of pulmonary surfactant, using a) surface tension measurement with the pulsating bubble surfactometer in suspensions and b) in surfactant films applying the hypophase exchanger. After hypophase exchange a preformed film of Survanta is very resistant to the inhibitory activity of alb or fib. The surface tensions of suspensions are significantly higher (p <0.001) than the surface tensions of preformed surfactant films if alb or fib were added, e.g., 42 (41 to 43) mN/m vs. 21 (19 to 22) mN/m for Survanta with 20 mg alb/ml. After additional supplementation of Survanta with SP-B the surface activity of Survanta/1% SP-B films did not show inhibition by fib (2 mg/ml), (surface tension 8 (4 to 13) mN/m). These results indicate that SP-B can play an important role to protect the pulmonary surfactant film from inactivation by serum proteins.

Importance of the carboxy-terminal 25 amino acid residues of lung collectins in interactions with lipids and alveolar type II cells.

Surfactant proteins A and D (SP-A and SP-D) are structurally related members of the collectin family found in the alveolar compartment of the lung. SP-A binds dipalmitoylphosphatidylcholine (DPPC) and galactosylceramide (GalCer), induces liposome aggregation, and regulates the uptake and secretion of surfactant lipids by alveolar type II cells in vitro. SP-D binds phosphatidylinositol (PI) and glucosylceramide. The purpose of this study was to identify a critical stretch of primary sequence in the SP-A region Cys(204)-Phe(228) and the SP-D region Cys(331)-Phe(355) that is involved in protein-specific lipid and type II cell interactions. Chimeras ad1 and ad2 were constructed with rat SP-A/SP-D splice junctions at Cys(218)/Gly(346) and Lys(203)/Cys(331), respectively. Chimera ad1 but not ad2 retained DPPC liposome binding activity. Both chimeras retained significant binding to GalCer liposomes. Chimera ad1 did not bind to PI, whereas chimera ad2 acquired a significant PI binding. Both chimeras failed to induce liposome aggregation and to interact with alveolar type II cells. In addition, monoclonal antibody 1D6 that blocks specific SP-A functions did not recognize either chimera. From these results, we conclude that (1) the SP-A region Leu(219)-Phe(228) is required for liposome aggregation and interaction with alveolar type II cells, (2) the SP-A region Cys(204)-Cys(218) is required for DPPC binding, (3) the SP-D region Cys(331)-Phe(355) is essential for minimal PI binding, and (4) the epitope for mAb 1D6 is located at the region contiguous to the SP-A region Leu(219)-Phe(228).

Antagonistic effects of pyrrolidine dithiocarbamate and N-acetyl-L-cysteine on surfactant protein A and B mRNAs.

Pulmonary surfactant, a mixture of phospholipids and specific associated proteins, reduces surface tension at the air-liquid interface of the lung and protects the large epithelial surface of the lung from infectious organisms. Surfactant proteins, SP-A and SP-B, are required for normal surfactant function. In the current work, increased levels of oxidized glutathione (GSSG) are demonstrated at doses of pyrrolidine dithiocarbamate (PDTC) which decrease SP-A and SP-B mRNAs, suggesting that cellular oxidation reduces surfactant protein expression. Similarly, reduction of SP-A and SP-B mRNA levels following accumulation of GSSG induced by glutathione reductase inhibitor 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), supports the hypothesis that surfactant protein synthesis is reduced in response to oxidation of pulmonary epithelial glutathione. Concurrent induction of apolipoprotein J (apoJ) mRNA by PDTC demonstrates the selectivity of pulmonary gene regulation by the dithiocarbamate. In contrast, the glutathione precursor N-acetyl-l-cysteine (NAC) prevented PDTC-dependent increase in GSSG/GSH ratio, inhibition of SP-A and -B mRNAs, and induction of apoJ. Insufficiency of SP-A and -B, which occurs in inflammatory lung diseases, may result from the exposure of the pulmonary epithelium to oxidant stress and may be reversed by the antioxidant NAC.

Introduction of mannose binding protein-type phosphatidylinositol recognition into pulmonary surfactant protein A.

Pulmonary surfactant protein A (SP-A) and mannose-binding protein A (MBP-A) are collectins in the C-type lectin superfamily. These collectins exhibit unique lipid binding properties. SP-A binds to dipalmitoyl phosphatidylcholine (DPPC) and galactosylceramide (GalCer) and MBP-A binds to phosphatidylinositol (PI). SP-A also interacts with alveolar type II cells. Monoclonal antibodies (mAbs PE10 and PC6) that recognize human SP-A inhibit the interactions of SP-A with lipids and alveolar type II cells. We mapped the epitopes for anti-human SP-A mAbs by a phage display peptide library. Phage selected by mAbs displayed the consensus peptide sequences that are nearly identical to 184TPVNYTNWYRG194 of human SP-A. The synthetic peptide GTPVNYTNWYRG completely blocked the binding of mAbs to human SP-A. Chimeric proteins were generated in which the rat SP-A region Thr174-Gly194 or the human SP-A region Ser174-Gly194 was replaced with the MBP-A region Thr164-Asp184 (rat ama4 or hu ama4, respectively). The mAbs failed to bind hu ama4. Rat ama4 bound to an affinity matrix on mannose-sepharose but lost all of the SP-A functions except carbohydrate binding and Ca2+-independent GalCer binding. Strikingly, the rat ama4 chimera acquired the PI binding property that MBP-A exhibits. This study demonstrates that the amino acid residues 174-194 of SP-A and the corresponding region of MBP-A are critical for SP-A-type II cell interaction and Ca2+-dependent lipid binding of collectins.

SP-A 3'-UTR is involved in the glucocorticoid inhibition of human SP-A gene expression.

The synthetic glucocorticoid dexamethasone has a major inhibitory effect on human surfactant protein A1 (SP-A1) and SP-A2 gene expression that occurs at both the transcriptional and posttranscriptional levels. Toward the identification of cis-acting elements that may be involved in the dexamethasone regulation of SP-A mRNA stability, chimeric chloramphenicol acetyltransferase (CAT) constructs that contained various portions of SP-A1 or SP-A2 cDNA in place of the native CAT 3'-untranslated region (UTR) were transiently transfected into the lung adenocarcinoma cell line NCI-H441. CAT activity was reduced in NCI-H441 cells by exposure to 100 nM dexamethasone only for the chimeric CAT constructs that contained the SP-A 3'-UTR. Moreover, the inhibitory response seen with dexamethasone was greater for the 3'-UTR derived from the SP-A1 allele 6A3 than with the 3'-UTR derived from either the SP-A1 allele 6A2 or SP-A2 allele 1A0, indicating differential regulation between SP-A genes and/or alleles.

The MAL proteolipid is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin in Madin-Darby canine kidney cells.

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.

C1qRP is a heavily O-glycosylated cell surface protein involved in the regulation of phagocytic activity.

C1q, mannose-binding lectin (MBL), and pulmonary surfactant protein A (SPA) interact with human monocytes and macrophages, resulting in the enhancement of phagocytosis of suboptimally opsonized targets. mAbs that recognize a cell surface molecule of 126,000 Mr, designated C1qRP, have been shown to inhibit C1q- and MBL-mediated enhancement of phagocytosis. Similar inhibition of the SPA-mediated enhancement of phagocytosis by these mAbs now suggests that C1qRP is a common component of a receptor for these macromolecules. Ligation of human monocytes with immobilized R3, a IgM mAb recognizing C1qRP, also triggers enhanced phagocytic capacity of these cells in the absence of ligand, verifying the direct involvement of this polypeptide in the regulation of phagocytosis. While the cDNA for C1qRP encodes a 631 amino acid membrane protein, Chinese hamster ovary cells transfected with the cDNA of the C1qRP coding region express a surface glycoprotein with the identical 126,000 Mr in SDS-PAGE as the native C1qRP. Use of glycosylation inhibitors, cleavage of the mature C1qRP with specific glycosidases, and in vitro translation of C1qRP cDNA demonstrated that both posttranslational glycosylation and the nature of the amino acid sequence of the protein contribute to the difference between its predicted m.w. and its migration on SDS-PAGE. These results verify that the cDNA cloned codes for the mature C1qRP, that C1qRP contains a relatively high degree of O-linked glycoslyation, and that C1qRP cross-linked directly by monoclonal anti-C1qRP or engaged as a result of cell surface ligation of SPA, as well as C1q and MBL, enhances phagocytosis.

Inhibition of hSP-B promoter in respiratory epithelial cells by a dominant negative retinoic acid receptor.

Retinoic acid (RA) receptors (RARs) belong to the nuclear hormone receptor superfamily and play important roles in lung differentiation, growth, and gene regulation. Surfactant protein (SP) B is a small hydrophobic protein synthesized and secreted by respiratory epithelial cells in the lung. Expression of the SP-B gene is modulated at the transcriptional and posttranscriptional levels. In the present work, immunohistochemical staining revealed that RAR-alpha is present on day 14.5 of gestation in the fetal mouse lung. To assess whether RAR is required for SP-B gene transcription, a dominant negative mutant human (h) RAR-alpha403 was generated. The hRAR-alpha403 mutant was transcribed and translated into the truncated protein product by reticulocyte lysate in vitro. The mutant retained DNA binding activity in the presence of retinoid X receptor-gamma to an RA response element in the hSP-B promoter. When transiently transfected into pulmonary adenocarcinoma epithelial cells (H441 cells), the mutant hRAR-alpha403 was readily detected in the cell nucleus. Cotransfection of the mutant hRAR-alpha403 repressed activity of the hSP-B promoter and inhibited RA-induced surfactant proprotein B production in H441 cells, supporting the concept that RAR is required for hSP-B gene transcription in vitro.

Glucose decreases steady state mRNA content of hydrophobic surfactant proteins B and C in fetal rat lung explants.

The streptozotocin-induced diabetic (STZ-DB) rat model is associated with fetal hyperglycemia, but with low to normal plasma insulin concentration. Because surfactant protein (SP) mRNA content in fetal rat lung is decreased in STZ-DB pregnancy, we investigated the effect of increasing concentrations of glucose on SP gene expression in lung organ cultures. SP mRNA content (SP-A, SP-B, SP-C) was assessed by Northern blot analysis in fetal day 20 lung explants (term = 22 days) cultured for 44 hours in medium containing 10, 25, 50, or 100 mM glucose. Our findings were (1) No consistent alteration in SP-A mRNA content was observed at different glucose concentrations (P > .05); (2) SP-B and SP-C mRNA content were reduced in a dose-dependent manner when glucose concentration was increased from 10 mM to 100 mM. The mRNA content, compared to 10 mM glucose, decreased to 50-60% at 25 mM glucose, to 20-25% at 50 mM glucose, and to lower than 10% at 100 mM glucose (P < .01). These findings indicate that the decrease in SP-B and SP-C mRNA in fetuses of STZ-DB rats may be, in part, due to a direct effect of hyperglycemia, whereas the decrease in SP-A mRNA content in STZ-DB rats appears to be due to other effects of diabetes in pregnancy.

Interaction of NK lysin, a peptide produced by cytolytic lymphocytes, with endotoxin.

NK lysin is a 9-kDa polypeptide that was originally isolated from porcine intestinal tissue based on its antibacterial activity. It is produced by cytolytic lymphocytes and is cytolytic against a number of different types of tumor cells. Here we report the binding of NK lysin to lipopolysaccharide (LPS) and its anti-LPS activity. NK lysin binds to matrix-coated LPS from Escherichia coli, Pseudomonas aeruginosa, and different strains of Salmonella enterica. Lipid A and polymyxin B inhibited the binding, demonstrating a preferential interaction of NK lysin with the lipid part of LPS. Chromium-labeled lymphoma cells were lysed by NK lysin, and LPS dose-dependently inhibited the cytolysis at equimolar amounts. In the same manner, NK lysin inhibited certain LPS-stimulated effects on mouse bone marrow cells as well as LPS binding to mouse granulocytes. These results suggest that NK lysin may be a another natural LPS-binding protein from lymphocytes that may participate in the endogenous defense response associated with elevated concentrations of LPS.