Rabbit Peritoneal Macrophages Research Articles

Immunodominant extracellular loops of Treponema pallidum FadL outer membrane proteins elicit antibodies with opsonic and growth-inhibitory activities.

The global resurgence of syphilis has created a potent stimulus for vaccine development. To identify potentially protective antibodies (Abs) against Treponema pallidum (TPA), we used Pyrococcus furiosus thioredoxin (PfTrx) to display extracellular loops (ECLs) from three TPA outer membrane protein families (outer membrane factors for efflux pumps, eight-stranded β-barrels, and FadLs) to assess their reactivity with immune rabbit serum (IRS). Five ECLs from the FadL orthologs TP0856, TP0858 and TP0865 were immunodominant. Rabbits and mice immunized with these five PfTrx constructs produced ECL-specific Abs that promoted opsonophagocytosis of TPA by rabbit peritoneal and murine bone marrow-derived macrophages at levels comparable to IRS and mouse syphilitic serum. ECL-specific rabbit and mouse Abs also impaired viability, motility, and cellular attachment of spirochetes during in vitro cultivation. The results support the use of ECL-based vaccines and suggest that ECL-specific Abs promote spirochete clearance via Fc receptor-independent as well as Fc receptor-dependent mechanisms.

Treponema pallidum recombinant protein Tp47 activates NOD-like receptor family protein 3 inflammasomes in macrophages via glycolysis

Glycolysis is a key pathway in cellular glucose metabolism for energy supply and regulates immune cell activation. Whether glycolysis is involved in the activation of NOD-like receptor family protein 3 (NLRP3) inflammasomes during Treponema pallidum (T. pallidum) infection is unclear. In this study, the effect of T. pallidum membrane protein Tp47 on NLRP3 inflammasome activation in rabbit peritoneal macrophages was analysed and the role of glycolysis in NLRP3 inflammasome activation was explored. The results showed that Tp47 promoted NLRP3, caspase-1, and IL-1β mRNA expression in macrophages, enhanced glycolysis and glycolytic capacity of macrophage, and promoted the production of macrophage glycolytic metabolites citrate, phosphoenolpyruvate, and lactate. The M2 pyruvate kinase (PKM2) inhibitor shikonin down-regulated the Tp47-promoted NLRP3, caspase-1, and IL-1β mRNA expression in macrophages, and suppressed the Tp47-enhanced glycolysis and glycolytic capacity. Similarly, si-PKM2 significantly inhibited Tp47-promoted NLRP3, caspase-1, and IL-1β mRNA expression and the Tp47-enhanced glycolysis and glycolytic capacity in macrophages. In conclusion, Tp47 activated NLRP3 inflammasomes via PKM2-dependent glycolysis and provided a new perspective on the effect of T. pallidum infection on host macrophages, which would contribute to the understanding of the infection mechanism and host immune mechanism of T. pallidum.

Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326).

Syphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Tp's repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of Tp by rabbit peritoneal macrophages. Three overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed "Epivolve" (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin scaffold (PfTrxBamA/ECL4). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (e.g., opsonization) and validate the mouse assay. Sera from Tp-infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using PfTrxBamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays. Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of PfTrxBamA/ECL4. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope. Epivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by Tp infection.

Global profiling of miRNA and protein expression patterns in rabbit peritoneal macrophages treated with exosomes derived from Taenia pisiformis cysticercus

Infection of Taenia pisiformis cysticercus is very frequently found in lagomorphs and causes serious economic losses to rabbit breeding industry. T. pisiformis cysticercus has evolved numerous strategies to manipulate their hosts. The release of exosomes is of importance in the interaction between host and parasite. However, the mechanism by which T. pisiformis cysticercus evades the host immune system for long-term survival within the host remains unclear. Using small RNA sequencing and TMT labelling proteomic, we profiled the expression patterns of miRNAs and proteins in rabbit peritoneal macrophages treated with T. pisiformis cysticercus exosomes. Seven differentially expressed (DE)-miRNAs and six DE-proteins were randomly selected to validate the accuracy of the sequencing data by qRT-PCR or western blot. Functions of DE-miRNAs and proteins were analyzed using public data bases. And DE-miRNAs–DE-proteins correlation network were established. CCK-8 assay was used to evaluate the effect of exosomes on macrophages proliferation. Cell cycle of macrophages, isolated from T. pisiformis-infected rabbits, was determined using flow cytometry. A total of 21 miRNAs were significantly differentially expressed, including three worm-derived miRNAs. The expressions of miRNAs and proteins were consistent with the sequencing results. DE-miRNAs targets were related to cell proliferation and apoptosis. Exosomes treatment resulted in a decrease of macrophages proliferation. In vivo, T. pisiformis cysticercus significantly induced S phase cell arrest. Moreover, DE-proteins were related to production of interferon-gamma and interleukin-12, and immunoregulation. Correlation network analysis revealed a negative correlation relationship between DE-miRNAs and DE-proteins. Among them, novel334 and tpi-let-7-5p have potential regulatory effects on IL1β and NFκB2 respectively, which imply that novel334–IL1β/tpi-let-7-5p–NFκB2 axis may be an important way that T. pisiformis cysticercus modulates host immune response through exosomes. Further understanding of these potential regulatory mechanisms will contribute to clarify the mechanism of escape mediated by T. pisiformis exosomes.

Evidence that immunization with TP0751, a bipartite Treponema pallidum lipoprotein with an intrinsically disordered region and lipocalin fold, fails to protect in the rabbit model of experimental syphilis.

Deconvolution of syphilis pathogenesis and selection of candidate syphilis vaccinogens requires detailed knowledge of the molecular architecture of the Treponema pallidum outer membrane (OM). The T. pallidum OM contains a low density of integral OM proteins, while the spirochete’s many lipoprotein immunogens are periplasmic. TP0751, a lipoprotein with a lipocalin fold, is reportedly a surface-exposed protease/adhesin and protective antigen. The rapid expansion of calycin/lipocalin structures in the RCSB PDB database prompted a comprehensive reassessment of TP0751. Small angle X-ray scattering analysis of full-length protein revealed a bipartite topology consisting of an N-terminal, intrinsically disordered region (IDR) and the previously characterized C-terminal lipocalin domain. A DALI server query using the lipocalin domain yielded 97 hits, 52 belonging to the calycin superfamily, including 15 bacterial lipocalins, but no Gram-negative surface proteins. Surprisingly, Tpp17 (TP0435) was identified as a structural ortholog of TP0751. In silico docking predicted that TP0751 can bind diverse ligands along the rim of its eight-stranded β-barrel; high affinity binding of one predicted ligand, heme, to the lipocalin domain was demonstrated. qRT-PCR and immunoblotting revealed very low expression of TP0751 compared to other T. pallidum lipoproteins. Immunoblot analysis of immune rabbit serum failed to detect TP0751 antibodies, while only one of five patients with secondary syphilis mounted a discernible TP0751-specific antibody response. In opsonophagocytosis assays, neither TP0751 nor Tpp17 antibodies promoted uptake of T. pallidum by rabbit peritoneal macrophages. Rabbits immunized with intact, full-length TP0751 showed no protection against local or disseminated infection following intradermal challenge with T. pallidum. Our data argue that, like other lipoprotein lipocalins in dual-membrane bacteria, TP0751 is periplasmic and binds small molecules, and we propose that its IDR facilitates ligand binding by and offloading from the lipocalin domain. The inability of TP0751 to elicit opsonic or protective antibodies is consistent with a subsurface location.

Membrane topology and cellular location of the Treponema pallidum glycerophosphodiester phosphodiesterase (GlpQ) ortholog.

Recent reports that isolated Treponema pallidum outer membranes contain an ortholog for glycerophosphodiester phosphodiesterase (GlpQ) (D. V. Shevchenko, D. R. Akins, E. J. Robinson, M. Li, O. V. Shevchenko, and J. D. Radolf, Infect. Immun. 65:4179-4189, 1997) and that this protein is a potential opsonic target for T. pallidum (C. E. Stebeck, J. M. Shaffer, T. W. Arroll, S. A. Lukehart, and W. C. Van Voorhis, FEMS Microbiol. Lett. 154:303-310, 1997) prompted a more detailed investigation of its physicochemical properties and cellular location. [14C]palmitate radiolabeling studies of a GlpQ-alkaline phosphatase fusion expressed in Escherichia coli confirmed the prediction from DNA sequencing that the protein is lipid modified. Studies using Triton X-114 phase partitioning revealed that the protein's amphiphilicity is due to lipid modification and that a substantial portion of the polypeptide is associated with the T. pallidum peptidoglycan sacculus. Three different approaches, i.e., (i) proteinase K treatment of intact treponemes, (ii) indirect immunofluorescence analysis of treponemes encapsulated in agarose beads, and (iii) opsonophagocytosis of treponemes incubated with antiserum against recombinant GlpQ by rabbit peritoneal macrophages, confirmed that GlpQ is entirely subsurface in T. pallidum. Moreover, rabbits hyperimmunized with GlpQ were not protected against intradermal challenge with virulent treponemes. Circular dichroism spectroscopy confirmed that the recombinant form of the polypeptide lacked discernible evidence of denaturation. Finally, GlpQ was not radiolabeled when T. pallidum outer membranes were incubated with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarene, a photoactivatable, lipophilic probe which promiscuously labels both proteins and lipids within phospholipid bilayers. Taken as a whole, these studies indicate that the T. pallidum GlpQ ortholog is a periplasmic protein associated predominantly with the spirochete's peptidoglycan-cytoplasmic membrane complex.

α-Tocopherol down-regulates scavenger receptor activity in macrophages

The aim of this study was to investigate the effect of α-tocopherol on scavenger receptor (SR) activity, SR class A (SR-A) mRNA expression and transcriptional regulation in macrophages. Scavenger receptor activity was determined quantitatively by uptake of DiI-acLDL (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein) in rabbit peritoneal macrophages and human monocytes/macrophages in the presence and absence of different tocopherol homologues. SR-A mRNA expression was determined by Northern blotting, the activity of the transcription factor activator protein-1 (AP-1) by electrophoretic mobility shift assay. We could demonstrate that α-tocopherol down-regulates scavenger receptor activity in macrophages in a dose-dependent manner. Scavenger receptor activity was reduced by 13, 16, 18 and 24% in the presence of 1, 5, 10 and 50 μM α-tocopherol, respectively. This effect was associated with a reduced SR-A mRNA expression and activity of AP-1 binding transcription factors in the presence of α-tocopherol. The activity of scavenger receptors in human monocyte derived macrophages incubated with 100 μM α-tocopherol for 15 days was reduced up to 60%. Interestingly, γ-tocopherol, which is a homologue of α-tocopherol with a comparable antioxidative capacity, showed only a weak suppression of SR activity, SR-A expression and AP-1 activity. Our observations point to the conclusion that the reduction of SR-A expression and activity in presence of α-tocopherol is possibly related to its direct action on cell signaling.

In vitro and in vivo effects of activated macrophage supernatant on distal limb wounds of ponies

Abstract Objective To determine whether monokines produced by activated rabbit peritoneal macrophages can inhibit development of exuberant granulation tissue formation in distal limb wounds in ponies. Design Randomized block. Animals 5 castrated male ponies, 2 to 6 years old and weighing 140 to 190 kg. Procedure In vitro activity of cell-free rabbit peritoneal macrophage supernatant was determined after incubation of fibroblasts from the flank and the distal portion of limbs of horses and ponies. Tritiated thymidine was then added, and after reincubation, radioactivity was measured. After creation of a 4-cm2, full-thickness wound on the mid dorsal aspect of each metacarpus and metatarsus of each pony, in vivo activity of the macrophage supernatant was evaluated. Biopsy specimens were collected at random sites near a border of each wound at 4, 6, and 10 weeks after creation of the wounds. Treatment effects were evaluated on the basis of presence of exuberant granulation tissue requiring excision, number of times that excision was required, total area of the wound, epithelialized area, area of granulation bed, and histologic evaluation of the biopsy specimens. Results The macrophage supernatant effectively inhibited proliferation of equine fibroblasts in vitro. No significant in vivo treatment effects were found among the 4 treatment groups. Conclusion and Clinical Relevance Monokines from stimulated rabbit peritoneal macrophages may have potential for improving wound healing in horses and ponies because of their effective inhibition in vitro of equine fibroblast proliferation. (Am J Vet Res 1996;57:1220-1224)

Dacron stimulation of macrophage transforming growth factor-β release

This study evaluated the effect of Dacron on the release of macrophage transforming growth factor-β (TGF-β), an endothelial cell growth inhibitor. Rabbit peritoneal macrophages were grown in minimum essential medium (MEM) with 10% fetal bovine serum (FBS) in the presence or absence of Dacron (0.5mm × 3mm particles). Media were collected three times each week for 7 weeks. For the TGF-β bioassay, mink lung epithelial cells (CCL64) were grown in MEM with 10% FBS. Test-conditioned media, 100 μl, were added ( n = 4), and incubated 48h. 3H-Thymidine ( 3H-TdR) uptake was determined and compared with 3H-TdR uptake using known pure TGF-β standards. Media samples were additionally pre-incubated with a neutralizing anti-TGF-β 1 antibody and the 3H-TdR uptake again quantitated. TGF-β activity in the conditioned media of macrophages exposed to Dacron exceeded the control media groups in all weeks, reaching significance ( P < 0.05) in weeks 3, 4, 5, 6 and 7. Pre-incubation of media samples with the anti-TGF-β 1 antibody inhibited this TGF-β activity in all weeks with statistical significance in weeks 1, 2, 3, 5 and 7. The inhibitory effects of Dacron on endothelialization may be explained by the Dacron-induced release of TGF-β from macrophages.

Differential response of normal and hypercholesterolemic rabbit peritoneal, splenic and alveolar macrophages to LPS stimulation

Differential response of normal and hypercholesterolemic rabbit peritoneal, splenic and alveolar macrophages to LPS stimulation

Monoclonal antibody to proteoglycan derived from Grifola frondosa (Maitake).

A murine monoclonal antibody (MAb) was prepared by immunizing BALB/c mice with a proteoglycan fraction derived from Grifola frondosa (Maitake mushroom), followed by the hybridization of spleen cells with mouse myeloma cells. The MAb (subclass; Ig G2b), designated MPG2, reacted with schizophyllan (SPG), curdlan, scleroglucan, laminarin and lentinan, but not with dextran, pullulan, mannan and xylan. Immunohistochemistry (ABC-GO method) showed that MAb MPG2 reacted with lysosomal proteoglycan and (1-->6)-beta-branched laminaritriose taken up by rabbit peritoneal macrophages. These results suggest that this MAb may recognize mainly (1-->3)-beta-D-glucan, and may be useful for determining the immunological properties of Grifola frondosa-derived proteoglycan.

Corticotropin-releasing hormone enhances the superoxide anion production of rabbit peritoneal macrophages stimulated with N-formyl-methionyl-leucyl-phenylalanine

Effects of corticotropin-releasing hormone (CRH) on the superoxide anion (O 2 −) production in macrophages (Møs) were investigated. CRH by itself failed to induce the (O 2 −) production of rabbit peritoneal Møs elicited with thioglycollate medium. However, Møs preincubated with CRH showed significant enhancement of (O 2 −) production following stimulation with a chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), at CRH concentrations from 10 −10 to 10 −7 M. Maximal enhancement of (O 2 −) production was obtained with 10 min preincubation with CRH for FMLP stimulation. CRH had no effect on (O 2 −) production when stimulated with phorbol 12-myristate 13-acetate. Neither corticotropin nor β-endorphin augmented the (O 2 −) production induced with FMLP. These results indicate that CRH has priming effects on FMLP-induced (O 2 −) production of rabbit peritoneal Møs, although the underlying mechanism remains to be clarified.

Exogenous glucocorticoids increase macrophage secretion of apo E by cholesterol-independent pathways

Macrophage apo E synthesis and secretion has been previously demonstrated to be regulated by intracellular free cholesterol levels and is decreased by cytokines and other inflammatory stimuli associated with macrophage activation. In a recent study, the opposing effects of TGFβ and GM-CSF were reported with the former increasing and the latter decreasing apo E secretion and apo E mRNA levels. In an attempt to further understand the mechanisms by which TGFβ increased apo E expression in mouse peritoneal macrophages, the present study was performed to determine whether pharmacological agents could up-regulate apo E secretion by a mechanism independent of intracellular free cholesterol levels. Agents which resulted in increased apo E secretion were subdivided based on their effects on cAMP elevation. In addition to TGFβ, dexamethasone resulted in significant increases in apo E secretion. The 2–4-fold enhancement in apo E secretion by both TGFβ and dexamethasone occurred without concomitant changes in intracellular cAMP or free cholesterol. Other agents which increased apo E secretion included cholera toxin and 8-bromocAMP. While these agents did not affect intracellular cholesterol levels, cholera toxin did increase macrophage cAMP. The changes in apo E secretion by dexamethasone and 8-bromo-cAMP were associated with elevations in apo E mRNA. Dexamethasone-treated macrophages had 6-fold increases in apo E mRNA by 48 h when compared with control macrophages. Macrophages stimulated with 8-bromo-cAMP for 48 h demonstrated a more modest but statistically significant ( P < 0.001) 2.2-fold increase. Similar effects of dexamethasone, cholera toxin, TGFβ, and 8-bromo-cAMP on apo E secretion were also apparent in macrophage-derived foam cells. In addition to increasing apo E secretion in macrophages and foam cells, dexamethasone and 8-bromo-cAMP inhibited the down-regulation of apo E secretion mediated by LPS and GM-CSF. Finally, the increased apo E secretion by exogenous glucocorticoids or TGFβ was not species specific as similar effects were observed in rabbit peritoneal macrophages. Therefore, while macrophage activation results in decreased apo E synthesis, macrophages exposed to anti-inflammatory agents including dexamethasone, TGFβ, or following cAMP elevation demonstrate increased apo E secretion by a cholesterol-independent mechanism.

Different expression of modified low density lipoprotein receptors in rabbit peritoneal macrophages and Kupffer cells

We have previously reported that mouse peritoneal macrophages have three types of modified low density lipoprotein (LDL) receptors. One is specific for acetylated LDL (Ac-LDL), the second is for oxidized LDL (Ox-LDL), and the third recognizes both (Arai, H. et al. (1989) Biochem. Biophys. Res. Commun. 159, 1375–1382). In the current study, the characteristics of modified LDL receptors in rabbit peritoneal macrophages and Kupffer cells from rabbits were investigated. Cross-competition studies of the degradation assay between Ox-LDL and Ac-LDL in rabbit peritoneal macrophages showed that the degradation of 125I-labeled Ox-LDL was almost completely inhibited by an excess amount of unlabeled Ac-LDL. On the other hand, an excess amount of unlabeled Ox-LDL suppressed 125I-labeled Ac-LDL degradation only partially. In contrast, in Kupffer cells an excess amount of unlabeled Ox-LDL inhibited the degradation of 125I-labeled Ac-LDL almost completely, whereas the degradation of 125I-labeled Ox-LDL was inhibited only partially by Ac-LDL. Scatchard analysis of binding assay showed that rabbit peritoneal macrophages have a single class of receptor for Ox-LDL, which binds maximally 0.31 μg/mg cellular protein ( B max ) with an apparent dissociation constant ( K d ) of 19.3 μg/ml, and two classes of receptors for Ac-LDL; one with high affinity ( B max 0.025 μg/mg cellular protein, K d 0.040 μg/ml) and the other with low affinity ( B max 0.08 μg/mg cellular protein, K d 11.31 μg/ml, On the other hand, Kupffer cells have two classes for Ox-LDL; one is a high affinity receptor ( B max 0.53 μg/mg cellular protein, K d 0.99 μg/ml) and the other is a low affinity receptor ( B max 3.71 μg/mg cellular protein, K d 16.2 μg/ml) and a single class for Ac-LDL ( B max 0.60 μg/mg cellular protein, K d 7.24 μg/ml). These results indicate that rabbit peritoneal macrophages have two kinds of modified LDL receptors; one is specific for Ac-LDL, and the other recognizes both Ox-LDL and Ac-LDL. These cells, thereby, have no specific receptor for Ox-LDL as was seen in mouse peritoneal macrophages. On the other hand, rabbit Kupffer cells have a receptor which recognizes Ox-LDL specifically. However, a specific receptor for Ac-LDL does not exist in rabbit Kupffer cells.

Thyrotropin-releasing hormone enhances the superoxide anion production of rabbit peritoneal macrophages stimulated with N-formyl-methionyl-leucyl-phenylalanine and opsonized zymosan

Effects of thyrotropin-releasing hormone (TRH) on the superoxide anion (O 2 −) production, which is essential for effective microbicidal and cytotoxic activity in macrophages (Mφs), were investigated. TRH by itself failed to induce the O 2 − production of rabbit peritoneal Mφs elicited with thioglycollate medium. However, Mφs preincubated with TRH showed the significant enhancements of O 2 − production following stimulation with the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), at TRH concentrations from 10 −7 to 10 −4 M with a peak enhancement at 10 −5 M. O 2 − generations of Mφs stimulated with opsonized zymosan (OZ) were also enhanced at TRH concentrations from 10 −10 to 10 −5 M with a peak enhancement at 10 −7 M. Maximal enhancements of O 2 − production were obtained with 10 min preincubation with TRH both for FMLP and OZ stimulations. TRH had no effect on O 2 − production when stimulated with phorbol 12-myristate 13-acetate. Thyrotropin did not augment the O 2 − production induced with either FMLP or OZ. These results indicate that TRH has the priming effect on FMLP- and OZ-induced O 2 − production of rabbit peritoneal Mφs, although the mechanism remains to be clarified.

Lipopolysaccharide binding protein enhances the responsiveness of alveolar macrophages to bacterial lipopolysaccharide. Implications for cytokine production in normal and injured lungs.

A plasma lipopolysaccharide (LPS)-binding protein (LBP) has been shown to regulate the response of rabbit peritoneal macrophages and human blood monocytes to endotoxin (LPS). We investigated whether LBP is present in lung fluids and the effects of LBP on the response of lung macrophages to LPS. Immunoreactive LBP was detectable in the lavage fluids of patients with the adult respiratory distress syndrome by immunoprecipitation followed by Western blotting, and also by specific immunoassay. In rabbits, the LBP appeared to originate outside of the lungs, inasmuch as mRNA transcripts for LBP were identified in total cellular RNA from liver, but not from lung homogenates or alveolar macrophages. Purified LBP enhanced the response of human and rabbit alveolar macrophages to both smooth form LPS (Escherichia coli O111B:4) and rough form LPS (Salmonella minnesota Re595). In the presence of LBP and LPS, the onset of tumor necrosis factor-alpha (TNF alpha) production occurred earlier and at an LPS threshold dose that was as much as 1,000-fold lower for both types of LPS. In rabbit alveolar macrophages treated with LBP and LPS, TNF alpha mRNA appeared earlier, reached higher levels, and had a prolonged half-life as compared with LPS treatment alone. Neither LPS nor LPS and LBP affected pHi or [Cai++] in alveolar macrophages. Specific monoclonal antibodies to CD14, a receptor that binds LPS/LBP complexes, inhibited TNF alpha production by human alveolar macrophages stimulated with LPS alone or with LPS/LBP complexes, indicating the importance of CD14 in mediating the effects of LPS on alveolar macrophages. Thus, immunoreactive LBP accumulates in lung lavage fluids in patients with lung injury and enhances LPS-stimulated TNF alpha gene expression in alveolar macrophages by a pathway that depends on the CD14 receptor. LBP may play an important role in augmenting TNF alpha expression by alveolar macrophages within the lungs.

Macrophage-Mediated Killing of Opsonized Treponema pallidum

The ability of proteose peptone-induced normal rabbit peritoneal macrophages to kill Treponema pallidum subspecies pallidum in vitro is demonstrated. Treponemes and 10% heated immune or normal sera were incubated with macrophages at a ratio of 1:200. After 2-10 h of incubation, these mixtures were injected intradermally at duplicate sites on normal rabbits. Maximal killing (failure to develop lesions) was seen at 10 h of incubation with immune serum: Only 7% (1/14) of lesions developed compared with 90% (9/10) after incubation in the presence of normal serum (P less than .001). Maximal phagocytosis (detected by immunofluorescence) occurred by 8 h in the presence of immune serum, when 90% of macrophages had ingested treponemes. At this point, however, 70% of lesion sites from macrophages incubated with treponemes and immune serum still developed, suggesting that effective killing may require at least 2 h after phagocytosis.

Rabbit Alveolar Macrophages Stimulated with Endotoxin and Lung Fragments from Endotoxemic Rabbits Produce a Leukocyte Infiltration-Inducing Factor That Lacks IL-1, TNFα, or Chemotactic Activity

We reported previously that rabbit pleural and peritoneal macrophages (Møs) and human Mø stimulated with endotoxin (LPS) release a protein factor of 45 to 60 kd that induces local polymorphonuclear leukocyte (PMNL) infiltration upon intradermal injection in rabbits. In the case of the human Mø product, it was shown to be distinct from interleukin-1 (IL-1), tumor necrosis factors (TNF alpha), granulocyte macrophage colony stimulating factor (GMCSF), IL-6, and lower molecular weight PMNL chemotactic factors. Here, we examined resident rabbit alveolar Møs to determine if they produce a similar factor following in vitro or in vivo exposure to LPS. Following LPS exposure (0.3 to 30 ng/ml), alveolar Møs obtained from normal rabbits by bronchoalveolar lavage released PMNL recruiting activity within 3 h, as measured by the accumulation of 51Cr labeled blood PMNL at injected skin sites. Production of this activity was blocked by cycloheximide; it was heat labile and not affected by polymyxin B, which neutralized the LPS. On gel filtration chromatography, a major peak of activity was eluted at 45 to 60 kd and was free of IL-1 but partially overlapped with rabbit TNF alpha. Although active in vivo in PMNL recruitment into the tissues, these fractions did not induce PMNL migration in vitro in a filter chemotaxis assay. After sodium dodecyl sulfate (SDS), polyacrylamide gel electrophoresis (PAGE) the predominant PMNL recruiting factor (PRF) eluted from gel slices corresponded to 22 to 32 kd, suggesting that this protein is a dimer under gel filtration conditions. These gel eluates did not contain TNF alpha activity. Following iv administration of sublethal doses of LPS (3 micrograms/kg) or of antibiotic killed Escherichi coli (10(9)/kg), peripheral lung fragments from perfused lungs spontaneously produced this PRF during ex vivo culture without further LPS stimulation. Lung tissue from normal rabbits did not release PRF spontaneously. We conclude that resident alveolar Møs produce a PRF protein in response to LPS that is distinct from IL-1, TNF alpha, and chemotactic factors and that the production of a similar protein by lung cells (probably Møs) is probably induced in vivo during endotoxemia or bacteremia. This factor may contribute to PMNL accumulation in the lung during pathologic processes.

Aortic endothelial cell damage induced by β-VLDL and macrophages in vitro

We performed an in vitro study to assess damage to swine aortic endothelial cells by rabbit β-VLDL and/or rabbit peritoneal macrophages. Incubation of cultured aortic endothelial cells with β-VLDL, macrophages, or macrophage lysate induced endothelial cell damage time- and dose-dependently as estimated by [ 3H]adenine release. Incubation of endothelial cells with both β-VLDL and macrophages produced a synergistic effect on the increase of [ 3H]adenine release. Pretreatment of the endothelial cells with some kinds of antioxidants (probucol 50 μg/ml, vitamin E 50 μM, superoxide dismutase-polyethylene glycol 0.5 mg/ml, or catalase-polyethylene glycol 0.5–1.0 mg/ml) significantly prevented the endothelial damage by β-VLDL or macrophage lysate. We conclude that β-VLDL and/or macrophages could induce endothelial cell damage and that some kinds of antioxidants could prevent it.

In vivo degradation of oxidized, regenerated cellulose

Oxidized, regenerated cellulose (ORC) was surgically implanted on the uterine horns of rabbits, and its biodegradation was studied in vivo. Samples of peritoneal lavages, serum, and urine were collected during the degradation process and analyzed for carbohydrate components utilizing high-performance liquid chromatography with pulsed amperometric detection (h.p.l.c.-p.a.d.). Degradation was rapid, and oligomeric products were evident primarily in the peritoneal fluid from the implantation site, with no apparent accumulation in either the serum or the urine. The size distribution and the amount of the oligomeric products decreased after day one, and by day four peritoneal lavages were essentially free of oligomers. The structure of the products formed was consistent with the lability of the polymer in solution, and the kinetics of degradation paralleled the results of the previously reported in vitro studies. Rabbit peritoneal macrophages, when incubated with ORC in vitro were observed to readily ingest and hydrolyze the polymeric material. A mechanism of degradation consisting of chemical depolymerization, followed by enzymatic hydrolysis mediated by glycosidases endogenous to peritoneal macrophages, is proposed.