Generation and characterization of hamster-mouse hybridomas secreting monoclonal antibodies with specificity for lipopolysaccharide receptor.

Experiments are described for the partial purification of the 80-kDa LPS binding protein expressed on macrophages and lymphocytes. This partially purified Ag was used to immunize adult Armenian hamsters and splenocytes from immunized animals were fused with murine myeloma cell lines. Hybridoma cell culture supernatants containing mAb were screened by ELISA for positive binding to the immunizing Ag, murine splenocytes and the murine 70Z/3 pre B cell and for an absence of binding to sheep E. Positive clones were further screened for reciprocal competitive binding with LPS on spleen cells and ability to modulate B lymphocyte mitogenic activity. Two hybridoma cell lines secreting IgM monoclonals, termed mAb3D7 and mAb5D3, were identified that satisfied all of the selection criteria. These hybridoma cell lines were subcloned and expanded. Binding of one (mAb3D7) was abrogated by treatment of Ag with mild periodate; binding of the second (mAb5D3) was destroyed by digestion of Ag with proteinase K. Binding specificity for mAb5D3 has been confirmed by ELISA using highly purified 80-kDa protein. These mAb have been of value in establishing that the 80-kDa LPS binding protein previously identified may serve as a specific functional receptor for LPS.     

Cytotoxic activity and production of toxic nitrogen oxides by macrophages treated with IFN-gamma and monoclonal antibodies against the 73-kDa lipopolysaccharide receptor.

The hamster IgM mAb 5D3 is specific for an 73-kDa LPS receptor on murine leukocytes. This mAb inhibits binding of radiolabeled LPS to splenocytes and acts as an agonist for induction of LPS-mediated changes in macrophage function. Resident peritoneal macrophages treated with IFN-gamma and mAb 5D3 developed potent cytotoxic activity against tumor cells. Cells treated with IFN-gamma or mAb 5D3 alone were inactive. Macrophage cytotoxic activity induced by IFN-gamma and mAb 5D3 was inhibited by NGMMLA and coincident with high levels of NO2-released into culture fluids. These data show that mAb 5D3 serves as an effective trigger signal for induction of cytotoxic activity with IFN-gamma-primed macrophages. Indeed, mAb 5D3 exactly mimicked the effects of LPS in these same systems. Unlike LPS, effects of mAb 5D3 on induction of macrophage cytotoxic activity and production of nitrogen oxides was abrogated after boiling, and not affected by addition of polymyxin B. The effects of LPS and mAb 5D3 as a trigger signal for IFN-gamma-primed macrophages were associated with production of TNF activity in culture fluids and inhibited by mAb against rTNF-alpha. Expression of class II MHC on macrophages induced by IFN-gamma treatment was suppressed by both LPS and mAb 5D3. These suppressive effects of LPS and mAb 5D3 were not affected by NGMMLA or mAb against rTNF-alpha. Finally, macrophages treated with LPS or mAb 5D3 before exposure to IFN-gamma and LPS or mAb 5D3 did not develop cytotoxic activity or high levels of NO2- in the culture fluids. These same cells developed both effector activities after addition of rTNF-alpha. These results in toto identify the 73-kDa protein as a receptor that mediates LPS-induced changes in macrophage effector function. The mAb 5D3 serves as a specific and defined reagent agonist for analysis of LPS receptor-linked change.     

Specific endotoxic lipopolysaccharide-binding proteins on murine splenocytes. I. Detection of lipopolysaccharide-binding sites on splenocytes and splenocyte subpopulations

Experiments have been carried out using a unique radio-iodinated, disulfide-reducible, photoactivatable LPS derivative (ASD-LPS) to detect specific LPS-binding proteins on murine splenocytes. Fractionation of LPS-photo-cross-linked, reduced, and solubilized splenocyte extracts on two-dimensional polyacrylamide gels has allowed the identification of an 80-kDa LPS-binding protein with approximate pI of 6.5. This LPS-binding protein is present on partially purified populations of splenic B lymphocytes, T lymphocytes, and macrophages. It is also the dominant LPS-binding protein on the murine 70Z/3 B cell line and the YAC-1 and EL4 T cell lines but is not detectable on the undifferentiated murine Sp2/0 myeloma cell line. Of potential importance is the fact that the 80-kDa protein appears to be indistinguishable when photolabeled extracts of splenocytes from the C3HeB/FeJ (lpsn) and LPS-nonresponder C3H/HeJ (lpsd) mice are compared.     

Specific endotoxic lipopolysaccharide-binding proteins on murine splenocytes. II. Membrane localization and binding characteristics

We have characterized the binding of LPS to an 80-kDa LPS-binding protein detected by an LPS photoaffinity probe to be present on murine splenocytes. Specific binding of LPS to the 80-kDa protein is directly proportional to LPS concentration at low concentrations of LPS and is saturable at high concentrations of LPS. Binding is inhibited by both homologous and heterologous underivatized LPS as well as by polysaccharide-free lipid A, indicating a specificity for the biologically active component of LPS. Analysis of the kinetics of binding indicate a time-dependent increase over the first 15 min, but increases are not detected after this time. Binding of LPS to the 80-kDa LPS-binding protein is reduced but still readily detectable at 4 degrees C in the presence of azide. The presence of the 80-kDa LPS-binding protein in an isolated cytoplasmic membrane fraction of murine splenocytes as well as its release from intact splenocytes by octylglucoside suggest that this LPS-binding protein is membrane localized. The results are consistent with, but do not establish unequivocally, the identity of the 80-kDa LPS-binding protein as a specific membrane receptor for lipid A.     

Heat shock protein 60: specific binding of lipopolysaccharide

Human heat shock protein 60 (HSP60) has been shown to bind to the surface of innate immune cells and to elicit a proinflammatory response. In this study we demonstrate that the macrophage stimulatory property of recombinant human HSP60 is tightly linked to the HSP60 molecule and is lost after protease treatment. However, inhibition of macrophage stimulation was reached by the LPS-binding peptide magainin II amide. Indeed, HSP60 specifically bound [(3)H]LPS. [(3)H]LPS binding to HSP60 was saturable and competable by the unlabeled ligand. To identify the epitope region of the HSP60 molecule responsible for specific LPS binding, we analyzed the effect of several anti-HSP60 mAbs on HSP60-induced production of inflammatory mediators from macrophages. We identified only one mAb, clone 4B9/89, which blocked the macrophage stimulatory activity of the chaperone. The epitope specificity of this mAb points to the region aa 335-366 of HSP60. Clone 4B9/89 also strongly inhibited [(3)H]LPS binding to HSP60. A more detailed analysis was performed by screening with selected overlapping 20-mer peptides of the HSP60 sequence, covering the region aa 331-380. Only one peptide blocked LPS binding to HSP60, thereby restricting the potential LPS-binding region to aa 351-370 of HSP60. Finally, analysis of selected 15-mer peptides and a 13-mer peptide of the HSP60 sequence revealed that most of the LPS-binding region was accounted for by aa 354-365 of HSP60, with the motif LKGK being critical for binding. Our studies identified a defined region of HSP60 involved in LPS binding, thereby implicating a physiological role of human HSP60 as LPS-binding protein.     

Products of lipopolysaccharide-activated macrophages (tumor necrosis factor-alpha, transforming growth factor-beta) but not lipopolysaccharide modify DNA synthesis by rat trophoblast cells exhibiting the 80-kDa lipopolysaccharide-binding protein

Pregnancy losses from gram negative bacterial infections could be caused by direct effects of LPS on placental cells, or indirectly via LPS activation of macrophages in the uteroplacental unit. To evaluate those alternatives, LPS, LPS-activated peritoneal cells, conditioned medium from LPS-activated peritoneal cells, and some purified and recombinant molecules known to be secreted by activated macrophages were tested for their abilities to modify DNA synthesis by rat trophoblast cells. Three trophoblast cell lines derived from midgestation placentas of outbred and inbred rats were used for the experiments. Although the 80-kDa LPS-binding protein was demonstrated on trophoblast cells, LPS alone had no effect on the ability of trophoblast cells to synthesize DNA. In cocultures, trophoblast cell DNA synthesis was slightly enhanced by low concentrations of both unstimulated and LPS-activated peritoneal cells. At higher concentrations, LPS-activated cells caused significant inhibition of DNA synthesis by trophoblast cells. Conditioned media from LPS-activated peritoneal cells were highly inhibitory to trophoblast cell DNA synthesis. When specific molecules likely to be components of those media were tested, IL-1 was found to have a modest but reproducible stimulatory effect and PGE2 did not change trophoblast cell incorporation of [3H]TdR. In contrast, trophoblast cell DNA synthesis was markedly inhibited in a dose-dependent manner by both TNF-alpha and TGF-beta 1. No differences in the sensitivity of trophoblast cells from outbred and inbred rats were observed. Given the limitations of the experimental model system, the results suggest that in cases of infection by gram-negative bacteria LPS may have an adverse effect on pregnancy by stimulating resident macrophages to generate and release molecules that are inhibitory to trophoblast cell DNA synthesis.     

Stimulation of macrophage tumoricidal activity by the growth and differentiation factor CSF-1

The effect of the macrophage growth and differentiation factor CSF-1 on the tumoricidal capacity of murine peritoneal exudate macrophages was investigated. Pretreatment of peptone-elicited macrophages 1 day with 300-1200 U/ml CSF-1 induced moderate killing and greatly stimulated lymphokine (LK)-induced killing of [3H]thymidine-labeled TU5 sarcoma cells to levels above that seen with fresh macrophages. Further addition of CSF-1 at Day 1 at the time of the tumor lysis assay promoted moderate increases in spontaneous and LK-induced activity. CSF-1 did not stimulate freshly harvested exudate macrophages to lyse TU5 targets in the presence or absence of lymphokine (LK) activators. Lipopolysaccharide (LPS) at 0.1-1000 ng/ml did not stimulate cytotoxicity, and the low endotoxin content and the use of polymyxin B and C3H/HeJ mice excluded a role for LPS in these experiments. Incubation of the macrophages with IFN and the myeloid growth factors IL-3 and GM-CSF did not stimulate tumoricidal activity. CSF-1 has been proposed as a therapeutic agent to restore myeloid cell numbers in induced (cancer chemotherapy, bone marrow transplantation, etc.) and natural aplastic anemias. These studies show that CSF-1 also may be useful in combination with LK activators to promote resistance to cancer in mature mononuclear cells. CSF-1 may have similar effects in LK-activated macrophages to enhance resistance to infectious diseases.     

Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14(+) cells.

Mammalian myeloid and epithelial cells express several kinds of antibacterial peptides (alpha-/beta-defensins and cathelicidins) that contribute to the innate host defense by killing invading micro-organisms. In this study we evaluated the LPS-neutralizing activities of cathelicidin peptides human CAP18 (cationic antibacterial proteins of 18 kDa) and guinea pig CAP11 using the CD14(+) murine macrophage cell line RAW264.7 and the murine endotoxin shock model. Flow cytometric analysis revealed that CAP18 and CAP11 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, Northern and Western blot analyses indicated that CAP18 and CAP11 suppressed LPS-induced TNF-alpha mRNA and protein expression by RAW264.7 cells. Interestingly, CAP18 and CAP11 possessed LPS-binding activities, and they strongly suppressed the interaction of LPS with LPS binding protein that mediates the transport of LPS to CD14 to facilitate the activation of CD14(+) cells by LPS. Moreover, when CAP18 and CAP11 were preincubated with RAW264.7 cells, they bound to the cell surface CD14 and inhibited the binding of FITC-LPS to the cells. Furthermore, in the murine endotoxin shock model, CAP18 or CAP11 administration inhibited the binding of LPS to CD14(+) cells (peritoneal macrophages) and suppressed LPS-induced TNF-alpha expression by these cells. Together these observations indicate that cathelicidin peptides CAP18 and CAP11 probably exert protective actions against endotoxin shock by blocking the binding of LPS to CD14(+) cells, thereby suppressing the production of cytokines by these cells via their potent binding activities for LPS and CD14.     

Taxol increases steady-state levels of lipopolysaccharide-inducible genes and protein-tyrosine phosphorylation in murine macrophages.

Taxol, a microtubule stabilizing agent, exhibits promise in the treatment of breast and ovarian tumors. Recently, this novel drug has been shown to activate murine macrophages to express TNF-alpha and to down-regulate TNF-alpha receptors, activities shared by bacterial LPS. Our study sought to determine if taxol could regulate gene expression in murine macrophages and to examine further the ability of taxol to generate an LPS-like signal. Toward this end, the ability of taxol to induce TNF-alpha mRNA and five other genes (IL-1 beta, IP-10, D3, D7, and D8) associated with LPS-activation of macrophages was examined by Northern blot analysis. Taxol alone (1-30 microM) induced murine C3H/OuJ macrophages to secrete bioactive TNF-alpha and express increased levels of each of the six genes under investigation. The magnitude and the kinetics of induction of each gene closely resembled that seen with Escherichia coli K235 LPS. Macrophages from LPS-hyporesponsive C3H/HeJ mice, however, failed to induce detectably any of the genes in response to taxol, despite being sensitive to the microtubule stabilizing effects of taxol as determined by immunofluorescence microscopy. The gene induction activity of taxol was in marked contrast to an alternative macrophage activator, heat killed Staphylococcus aureus, which induced a distinct gene profile in C3H/OuJ macrophages and which was equally active in C3H/OuJ and C3H/HeJ macrophages. These data are consistent with an ability of taxol to generate an LPS-like signal, possibly through a common signaling intermediate. As a first step toward identifying signal responses shared by taxol and LPS, we have shown that taxol, as shown previously for LPS, rapidly induces the tyrosine phosphorylation of a 41- and 42-kDa protein.     

Cellular and metabolic requirements for induction of macrophage procoagulant activity by murine hepatitis virus strain 3 in vitro.

The cellular basis for the variation in induction of monocyte procoagulant activity (PCA) by murine hepatitis virus strain 3 (MHV-3) was examined using a set of recombinant inbred strains of mice derived from the resistant (A/J) and susceptible C57B1/6J (B) progenitors. Induction of PCA by MHV-3 required live virus and host protein and RNA synthesis. Absolute restriction for induction of PCA was observed at the level of the macrophage. Peritoneal macrophages from resistant parental A/J and RI strains (AXB5) could not be induced to express PCA when stimulated by MHV-3 alone or in the presence of lymphocytes from susceptible and H-2 compatible RI mice (AXB3) although they did respond to endotoxin (LPS). In contrast, macrophages from both susceptible (AXB3) and semisusceptible (AXB1) RI strains of mice expressed a similar increase in PCA after stimulation with MHV-3 in the absence of lymphocytes. The levels of PCA expressed by macrophages in the presence of Thy-1.2+ lymphocytes correlated with susceptibility to disease. Thy-1.2+ lymphocytes from susceptible RI AXB3 mice could induce levels of PCA in macrophages from semisusceptible RI AXB1 mice equivalent to that seen in cultures of macrophages and lymphocytes from susceptible mice. Further subfractionation of Thy-1.2+ cells demonstrated that L3T4+ cells instructed macrophages to produce PCA. Thy-1.2+ cells from MHV-3 immunized resistant AXB5 mice, but not from non-immunized mice, were able to suppress induction of PCA. This suppressor cell activity could be detected 4 days after immunization, reaching maximal activity at day 7 with significant suppression even at 28 days. The PCA was shown to have direct prothrombin cleaving activity (prothrombinase) by ELISA and immunofluorescence staining using the mAb 3D4.3. These results demonstrate that induction of a unique PCA (prothrombinase) is restricted at the level of the macrophage and define a regulatory role for T lymphocytes in its induction.     

Specific endotoxic lipopolysaccharide-binding receptors on murine splenocytes. III. Binding specificity and characterization.

In previously published studies, we employed a photoreactive radioiodinated derivative of LPS from Escherichia coli 0111:B4 to identify and characterize a membrane-localized specific LPS binding protein of approximately 80-kDa molecular mass. Our more recent studies demonstrating that mAb with specificity for this 80-kDa protein will act as an agonist in mediating macrophage activation have established that this protein serves as a specific receptor for LPS. In the experiments reported here, we have more accurately determined the apparent molecular mass of this protein to be 73 kDa (p73). We have also extended the sources of LPS-derivatized photo-cross-linking preparations (including Re-LPS) to determine generality of LPS binding to this receptor. Binding to the p73 LPS receptor is demonstrated with all of the LPS derivatives synthesized in our laboratory, as well as probes synthesized by other investigators. Binding of S-LPS is readily inhibited by Re chemotype LPS, and we have shown that this competitive inhibition is most likely not the result of formation of LPS aggregates. These results confirm and extend our earlier studies suggesting that the binding of LPS to the p73 receptor is lipid A specific. We further demonstrate that, in contrast to results published in a recent report, the p73 LPS receptor has no significant binding specificity for a variety peptidoglycan polymer preparations. Finally, we show that this LPS receptor can be detected on murine fibroblast, macrophage, and mastocytoma cell lines. Differences have been observed in the level of expression of LPS receptors on the various cell lines studied.     

Lipopolysaccharide receptor on rabbit peritoneal macrophages. I. Binding characteristics

The Bordetella pertussis endotoxin, labeled with tritium ((3H)-LPS), bound irreversibly and nonspecifically to rabbit lung macrophages, but bound reversibly and specifically to both resident and elicited rabbit peritoneal macrophages. The specific binding capacity of the macrophages was saturated with about 3 X 10(4) LPS molecules per cell. The binding was inhibited with the homologous unlabeled endotoxin, but not at all with endotoxin from Proteus mirabilis, thus assessing ligand specificity. Endotoxins from other bacteria gave intermediate inhibition value. Binding of tritium-labeled pertussis endotoxin was significantly inhibited by one of the two polysaccharides (PS-1) present in this endotoxin, but neither the other polysaccharide (PS-2) nor the Lipid A fragment exhibited such activity. These results strongly suggest the presence of a lectin-like receptor for LPS on the membrane of rabbit peritoneal macrophages.     

Histones: a novel class of lipopolysaccharide-binding molecules

Unlike soluble and membrane forms of lipopolysaccharide (LPS)-binding proteins, intracellular LPS-binding molecules are poorly documented. We looked for such molecules in a murine lung epithelial cell line. Two proteins with LPS-binding activity were isolated and unambiguously identified as histones H2A.1 and H4 by mass spectrometry. Synthetic peptides representing partial structures indicated that the LPS binding site is located in the C-terminal moiety of the histones. Extending the study, we found that histones H1, H2A, H2B, H3, and H4 from calf thymus are all able to bind LPS. Bindings were specific, and affinities, determined by isothermal titration calorimetry, were (except for H4) higher than that of the LPS-binding antibiotic polymyxin B. In the presence of H2A the binding of LPS to the macrophage cell line RAW 264.7, and the LPS-induced production of TNF-alpha and nitric oxide by these cells, were markedly reduced. Histones may thus represent a new class of intracellular and extracellular LPS sensors.     

Development and characterization of monoclonal antibodies to murine macrophage colony-stimulating factor

The macrophage-specific CSF (CSF-1), purified from murine L cell-conditioned medium, supports the in vitro proliferation and survival of various murine mononuclear phagocyte colony-forming cells. In this report we describe the production and functional characterization of two monoclonal antibodies (mAb) to CSF-1 obtained from rat X rat hybridomas. These two mAb are functionally distinct and recognize different epitopes on CSF-1. The mAb 5A1 binds to and inhibits the biologic function of CSF-1, and the second mAb (D24) binds CSF-1 but does not neutralize its biologic activity. The mAb 5A1 inhibits colony formation of tissue mononuclear phagocyte colony-forming cells as well as the committed bone marrow stem cells for both granulocytes and monocytes. The extent of colony inhibition by mAb 5A1 is dependent on the tissue origin of colony-forming cells. CSF-1 complexed with mAb 5A1 does not bind to its cell surface receptor of peritoneal exudate macrophages, and mAb 5A1 does not complex with cell-bound CSF-1. Although both bone marrow cell-derived macrophages and J774.1 macrophages bind CSF-1, mAb 5A1 inhibits the proliferation of only bone marrow cell-derived macrophages. The non-neutralizing mAb D24 does not block binding of CSF-1 to its cellular receptor, and it recognizes cell-bound CSF-1.     

Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2

Toll-like receptor (TLR) 2 has recently been associated with cellular responses to numerous microbial products, including LPS and bacterial lipoproteins. However, many preparations of LPS contain low concentrations of highly bioactive contaminants described previously as "endotoxin protein," suggesting that these contaminants could be responsible for the TLR2-mediated signaling observed upon LPS stimulation. To test this hypothesis, commercial preparations of LPS were subjected to a modified phenol re-extraction protocol to eliminate endotoxin protein. While it did not influence the ability to stimulate cells from wild-type mice, repurification eliminated the ability of LPS to activate cells from C3H/HeJ (Lpsd) mice. Additionally, only cell lines transfected with human TLR4, but not human or murine TLR2, acquired responsiveness to both re-extracted LPS and to a protein-free, synthetic preparation of lipid A. These results suggest that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminating endotoxin protein.     

An anti-endotoxin peptide that generates from the amino-terminal domain of complement regulatory protein C1 inhibitor

C1 inhibitor (C1INH), a complement regulatory protein, prevents endotoxin shock via a direct interaction of the amino-terminal domain with gram-negative bacterial lipopolysaccharide (LPS). Importantly, the cleaved, inactive C1INH still is an anti-endotoxin effector indicating the anti-endotoxin peptide that generates from the amino-terminal domain of C1INH. In this study, we first identified that a cleaved fragment within the major part of the amino-terminal domain in in vitro proteolytic analysis of C1INH had an ability to bind to LPS. We synthesized several peptides overlapping the C1INH cleaved fragment. Among these synthetic peptides, a 13-mer derivative peptide at position from 18 to 30, named N2((18-30)), exhibited the most powerful anti-endotoxin activity in vitro, enlightening that it was most strong at binding to LPS, inhibiting the interaction of LPS with LPS-binding protein (LBP), blocking LPS binding to CD14(+) cells, and suppressing production of tumor necrosis factor (TNF)-alpha by murine macrophages, RAW 264.7. In the murine endotoxin shock model, the peptide N2((18-30)) protected mice from LPS-induced lethal septic shock by inhibiting macrophage activation. These data indicate that the peptide N2((18-30)) derived from the amino-terminal region of C1INH is anti-endotoxin.     

Peptidoglycan and lipopolysaccharide bind to the same binding site on lymphocytes

Bacterial cell wall peptidoglycan (PGN) and lipopolysaccharide (LPS), which are both macrophage activators and polyclonal B cell mitogens, were shown to bind to the same dominant 70-kDa 6.5 pI protein on the surface of mouse B lymphocytes. This conclusion was supported by the following results: (a) the PGN- and LPS-binding proteins co-migrated following photoaffinity cross-linking and two-dimensional polyacrylamide gel electrophoresis; (b) cross-linking of PGN to this 70-kDa protein was competitively inhibited by LPS (IC50 = 7.3 microM), LPS from a deep rough mutant (IC50 = 6.9 microM), and lipid A (IC50 = 18-72 microM); (c) cross-linking of LPS to this 70-kDa protein was competitively inhibited by polymeric soluble PGN (IC50 = 0.09 microM) and sonicated high Mr PGN (IC50 = 0.6 microM); (d) cross-linking of both PGN and LPS to this 70-kDa protein was also competitively inhibited by dextran sulfate (IC50 = 115-124 microM); (e) cross-linking of both PGN and LPS to this 70-kDa protein was inhibited by a (GlcNAc)2-specific lectin; and (f) peptide maps of the 70-kDa proteins digested with chymotrypsin, subtilisin, staphylococcal protease V, or papain were identical for PGN- and LPS-binding proteins and unique for each enzyme. Based on competitive inhibition experiments, binding of PGN to the 70-kDa protein was 20-1200 times stronger than the binding of LPS or lipid A on a per mol basis. However, when aggregated micellar structures of LPS or lipid A were considered, the avidities of LPS and PGN binding were similar. These results demonstrate binding of PGN and LPS to the same 70-kDa protein on lymphocytes and suggest that the binding is specific for the (GlcNAc-MurNAc)n backbone of PGN and the (GlcNAc)2 part of lipid A.