Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer.

Apoptosis is an evolutionarily conserved and tightly regulated cell death modality. It serves important roles in physiology by sculpting complex tissues during embryogenesis and by removing effete cells that have reached advanced age or whose genomes have been irreparably damaged. Apoptosis culminates in the rapid and decisive removal of cell corpses by efferocytosis, a term used to distinguish the engulfment of apoptotic cells from other phagocytic processes. Over the past decades, the molecular and cell biological events associated with efferocytosis have been rigorously studied, and many eat-me signals and receptors have been identified. The externalization of phosphatidylserine (PS) is arguably the most emblematic eat-me signal that is in turn bound by a large number of serum proteins and opsonins that facilitate efferocytosis. Under physiological conditions, externalized PS functions as a dominant and evolutionarily conserved immunosuppressive signal that promotes tolerance and prevents local and systemic immune activation. Pathologically, the innate immunosuppressive effect of externalized PS has been hijacked by numerous viruses, microorganisms, and parasites to facilitate infection, and in many cases, establish infection latency. PS is also profoundly dysregulated in the tumor microenvironment and antagonizes the development of tumor immunity. In this review, we discuss the biology of PS with respect to its role as a global immunosuppressive signal and how PS is exploited to drive diverse pathological processes such as infection and cancer. Finally, we outline the rationale that agents targeting PS could have significant value in cancer and infectious disease therapeutics.

Intratracheal administration of interleukin 12 plasmid-cationic lipid complexes inhibits murine lung metastases.

Administration of plasmid/lipid complexes to the lung airways for the treatment of metastatic pulmonary diseases represents a new strategy of gene therapy. In this study we present evidence that intratracheal administration of a plasmid encoding murine IL-12 complexed with N-[1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride:cholesterol inhibits the growth of lung metastases, using a renal cell carcinoma model. Instillation of pIL-12/lipid complexes resulted in expression of biologically active IL-12 (170-240 pg/ml) and IFN-gamma (100-190 pg/ml) in the bronchoalveolar lavage fluid. A significantly reduced number of lung metastases (26+/-24) was observed in mice instilled with IL-12/lipid complexes 24 hr after tumor challenge, whereas more than 250 metastatic foci were counted in lungs of untreated mice. Moreover, IL-12/lipid inhibited the growth of 3-day-old established metastases when compared with empty plasmid/lipid or IL-12 plasmid in saline. Mice receiving IL-12 gene therapy survived significantly longer (median survival of 43 days) than untreated mice (median survival of 31 days) or mice treated with control plasmid/lipid complexes (median survival of 35 days). These data demonstrate that a nonviral IL-12 gene therapy employing synthetic cationic lipids as a delivery system can be used to inhibit the development of lung metastases. Thus, this method provides support for the use of IL-12/lipid complexes to control the growth of pulmonary metastases and represents a potentially safer alternative to IL-12 protein immunotherapy.

Ultrasonic nebulization of cationic lipid-based gene delivery systems for airway administration.

PURPOSE: This study relates to the development of gene therapies for the treatment of lung diseases. It describes for the first time the use of ultrasonic nebulization for administration of plasmid/lipid complexes to the lungs to transfect lung epithelial cells. METHODS: Plasmid complexed to cationic liposomes at a specific stoichiometric ratio was nebulized using an ultrasonic nebulizer. We assessed: (i) the stability of plasmid and plasmid/lipid complexes to ultrasonic nebulization, (ii) the in vitro activity of plasmid in previously nebulized plasmid/lipid complex, (iii) the in vivo transgene expression in lungs following intratracheal instillation of nebulized plasmid/lipid formulations compared to un-nebulized complexes, (iv) the emitted dose from an ultrasonic nebulizer using plasmid/lipid complexes of different size, and (v) the transgene expression in lungs following oral inhalation of aerosolized plasmid/lipid complex generated using an ultrasonic nebulizer. RESULTS: Integrity of plasmid formulated with cationic lipids, and colloidal stability of the plasmid/lipid complex were maintained during nebulization. In contrast, plasmid alone formulated in 10% lactose was fragmented during nebulization. The efficiency of transfection of the complex before and after nebulization was comparable. Nebulization produced respirable aerosol particles. Oral exposure of rodents for 10 minutes to aerosol produced from the ultrasonic nebulizer resulted in transgene expression in lungs in vivo. CONCLUSIONS: The performance characteristics of the ultrasonic nebulizer with our optimized plasmid/lipid formulations suggests that this device can potentially be used for administering gene medicines to the airways in clinical settings for the treatment of respiratory disorders.

Target specific optimization of cationic lipid-based systems for pulmonary gene therapy.

PURPOSE: Cationic lipids are capable of transferring foreign genes to the pulmonary epithelium in vivo. It is becoming increasingly clear that factors other than lipid molecular structure also influence efficiency of delivery using cationic lipid systems. This study is aimed at evaluating the effect of formulation variables such as cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and plasmid topology on transgene expression in the lung. METHODS: The effect of varying the surface and colloidal properties of cationic lipid-based gene delivery systems was assessed by intratracheal instillation into rats. An expression plasmid encoding chloramphenicol acetyl transferase (CAT) was used to measure transgene expression. RESULTS: Cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and topology of the plasmid, were found to significantly affect transgene expression. Complexation with lipids was found to have a protective effect on DNA integrity in bronchoalveolar lavage fluid (BALF). DNA complexed with lipid showed enhanced persistence in rat lungs as measured by quantitative polymerase chain reaction. CONCLUSIONS: Fluorescence microscopy analysis indicated that the instilled formulation reaches the lower airways and alveolar region. Data also suggests cationic lipid-mediated gene expression is primarily localized in the lung parenchyma and not infiltrating cells isolated from the BALF.

Cationic lipids enhance cytokine and cell influx levels in the lung following administration of plasmid: cationic lipid complexes.

Administration of plasmid/lipid complexes to the lung airways may be associated, in addition to expression of transgene, with a range of other responses. We report here the induction of cytokines and cellular influx in the lung airway following intratracheal administration of an N-[1-(2-3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/cholesterol/plasmid positively charged complex in mice. We show that 1) the appearance of the Th1-associated cytokines IFN-gamma and IL-12 in bronchoalveolar lavage fluid is caused by unmethylated CpG dinucleotide sequences present within the plasmid, and is enhanced by the lipid formulation; 2) cationic lipids by themselves do not induce IL-12 or IL-12p40; 3) TNF-alpha is rapidly induced by cationic lipids and plasmid/lipid complex, but not by plasmid alone; 4) an acute cellular influx is induced by cationic lipid alone and by a plasmid/lipid complex, but to a much lesser extent by plasmid alone; and 5) plasmid methylation does not influence the degree of inflammatory cell influx. The induction of the innate immune responses by plasmid/lipid complexes may be advantageous to gene therapy of lung diseases. In particular, induction of the Th1 cell-promoting cytokines by plasmid/lipid complexes could, in conjunction with an expressed transgene, be used to modulate immune responses in the lung airways in disease conditions that are deficient in Th1 cell responses or that have a dominant Th2 phenotype. Alternatively, the elimination of immunostimulatory sequences in plasmids may improve the tolerability and/or efficacy of nonviral gene therapy, especially for diseases requiring chronic administration.

Isothiazolones interfere with normal matrix metalloproteinase activation and inhibit cartilage proteoglycan degradation.

A series of isothiazolones that inhibit pro-(matrix metallo-proteinase) (proMMP) activation but do not inhibit the active enzyme are effective as cartilage protectants in bovine nasal cartilage organ culture, preventing interleukin-1 (IL-1)-induced proteoglycan (aggrecan) degradation without affecting its synthesis. These compounds were found to bind to prostromelysin (proMMP-3) in a non-dialysable and stoichiometric manner. Preincubation with cartilage-protectant isothiazolones prevented the binding of [14C]iodoacetamide to Cys75 of the MMP-3 propeptide, suggesting that the activity of these compounds involves their binding to the Cys75 of the MMP zymogen. Studies following chymotrypsin activation of proMMP-3 by SDS/PAGE indicated that altered processing of the 57 kDa zymogen to the active form occurred in the presence of compound. The 53 kDa intermediate seen on normal activation was not formed; instead a different intermediate appeared with a molecular mass of approx. 46 kDa. N-terminal sequence analysis indicated that this intermediate was formed by cleavage at the putative 4-aminophenylmercuric acid cleavage site. Importantly the 45 kDa active MMP-3 species formed in the presence of compound was one amino acid residue shorter than the native MMP-3. These results suggest that the inhibition of cartilage proteoglycan degradation by isothiazolones might be due to their ability to bind to the Cys75 in the propeptide region of the MMP zymogen and interfere with its normal activation process.

2,5-Diarylisothiazolone: novel inhibitors of cytokine-induced cartilage destruction.

A series of 2,5-diarylisothiazolones is reported that inhibit the IL-1 beta-induced breakdown of bovine nasal septum cartilage in an organ culture assay. The synthesis and preliminary SAR of these compounds are described. These compounds represent a novel, nonpeptide lead series approach to the mediation of the chronic cartilage breakdown associated with arthritic disease. These compounds are relatively resistant to reductive metabolism by liver microsomal preparations and appear to inhibit cartilage breakdown by interfering with the proteolytic activation of matrix metalloproteinases.

Metabolism resistant isothiazolone inhibitors of cartilage breakdown.

A series of 2-(arylmethyl)pyridoisothiazolones is reported that inhibit the IL-1 beta induced breakdown of bovine nasal septum cartilage in an organ culture assay. The synthesis and preliminary SAR of these compounds are described. These compounds represent a novel, non-peptide lead series approach to the mediation of the chronic cartilage breakdown associated with arthritic disease. These compounds are relatively resistant to reductive metabolism by liver microsomal preparations and appear to inhibit cartilage breakdown by interfering with the proteolytic activation of matrix metalloproteinases.

Multiple sites of the propeptide region of human stromelysin-1 are required for maintaining a latent form of the enzyme.

Latency of matrix metalloproteinase 3 (MMP-3) is regulated by the interaction of a free cysteine residue (Cys-75) in the conserved amino acid sequence Pro-Arg-Cys-Gly-Val-Pro-Asp located in the COOH-terminal portion of the propeptide with a chelated zinc atom in the active site of the catalytic domain. Proteolytic activation of full-length human pro-MMP-3 involves the removal of approximately 35 amino acids from the NH2-terminal portion of the propeptide, forming a 53-kDa unstable intermediate that undergoes intermolecular autocatalysis to form the 45-kDa mature active enzyme. In this study, we have evaluated the contribution of the NH2-terminal 35 amino acids to the maintenance of latency. Full-length human pro-MMP-3 was expressed in Escherichia coli and refolded to form latent pro-MMP-3 capable of activation by chymotrypsin or aminophenylmercuric acetate. Renaturation of pro-MMP-3 expressed in bacteria with 20 or more amino acids removed from the NH2-terminal region of the propeptide yielded only an active enzyme. COS-7 cells transiently transfected with pro-MMP-3 expression vectors containing the single amino acid substitutions Y20A, L21A, and C75S also secreted active forms of the enzyme. These data suggest that simultaneous interactions of the NH2- and COOH-terminal regions of the propeptide are required for maintenance of the latent form of the enzyme.

Heteroaryl-fused 2-phenylisothiazolone inhibitors of cartilage breakdown.

The synthesis, biological evaluation, and structure-activity relationships of a series of N-phenyl heteroaryl-fused isothiazolones are described. These isothiazolones have been shown to exhibit potent, dose-dependent inhibition of IL-1 beta-induced breakdown of proteoglycan in a cartilage organ culture assay. This effect is likely due to inhibition of MMP activation and a consequent reduction in MMP activity following IL-1 beta stimulation. Thus these compounds potentially represent simple, non-peptidic disease-modifying agents for the treatment of arthritic diseases. To examine the effects of structure on in vitro activity, three general features of the molecules were varied, substituents on the pendant N-phenyl group, the position of ring fusion to the isothiazolone, and substituents on the fused ring peri to the isothiazolone sulfur.

Production of human matrix metalloproteinase 3 (stromelysin) in Escherichia coli.

Full-length human matrix metalloproteinase 3 (prostomelysin or proMMP-3) was produced in Escherichia coli as an intracellular insoluble aggregate that could be solubilized and refolded to yield an activatable proenzyme. The refolded protein was purified to > 95% homogeneity. The recombinant proMMP-3 (re-proMMP-3) could be activated by agents known to stimulate self-catalyzed cleavage of native fibroblast proMMP-3. The N-terminal amino-acid sequence of the re-proMMP-3 and its activation products indicated that they were the same as those obtained with the natural material.

IL-1 and its role in rat carrageenan pleurisy.

The carrageenan pleurisy model, which is characterized by cellular influx and oedema, has been used to examine the effects of anti-inflammatory compounds such as naproxen. Interleukin-1alpha and beta (IL-1) are known to be pro-inflammatory mediators, and their roles in this model are unknown. Intrapleural injection of 1% viscarin carrageenan or saline was administered to male Lewis rats. Four to 24 h later, cell counts, fluid volumes and IL-1beta levels (measured by ELISA) were determined in the pleural cavity. Serum corticosterone levels were measured only at 4 h. Significant increases in IL-1beta levels precede cell influx suggesting IL-1beta plays a role in the maintenance of cell accumulation in the pleural cavity. None of the drugs tested, including the IL-1 receptor antagonist, maintained pleural cell influx and IL-1beta levels at control levels. When human IL-1alpha or beta or rat IL-1beta were injected individually into the pleural cavity, none of these cytokines were pro-inflammatory, as measured by increased cell influx and fluid extravasation. These results suggest that although IL-1beta levels increase in the pleural cavity in response to carrageenan, IL-1 per se is not the initiator of the pro-inflammatory events of cell influx and oedema in this model.

Quantitative characterization of the intrinsic ligand-binding affinity of the interleukin 2 receptor beta chain and its modulation by the alpha chain and a second affinity-modulating element.

The interleukin 2 receptor is a multisubunit receptor known to consist of at least two IL-2 binding subunits, alpha and beta. We report here kinetic evidence defining the contribution of an affinity-modulating element(s) intimately involved in modulation of the ligand-binding affinity of the beta chain and alpha/beta complex. The principal effect of this modulating element on the beta chain is to slow the dissociation of IL-2 more than 150-fold and thus raise its low intrinsic IL-2 binding affinity (Kd = 70 nM) as defined in transfected fibroblast cells to the level observed in lymphoid cells (Kd = 1.2 nM). The alpha subunit also increases the ligand-binding affinity of the beta chain, although in this case principally by increasing the association rate constant more than 1200-fold. The additional effect of the affinity-modulating element on the alpha/beta complex is minimal with regards to the equilibrium binding affinity. It does, however, have a detectable 14-fold effect on slowing the IL-2 dissociation rate. The existence of multiple forms of IL-2 receptor complexes with widely varying ligand affinities and dissociation rates illustrates the need for careful evaluation of binding data in studies of receptor subunit composition and reconstitution.

Functional expression of native and chimeric human and murine IL-2 receptor p70 chains in an IL-3-dependent murine cell line.

Full length cDNAs encoding the human and murine p70 genes were isolated using polymerase chain reaction (PCR) and conventional cDNA library screening techniques, respectively. To validate their functional potential, expression vectors containing human, murine and chimeric human/murine p70 cDNAs were transfected into the murine IL-3-dependent cell line FDC-P1. Transfected cells expressed a combination of high and low-affinity IL-2 binding sites while parental cells displayed only the low-affinity sites associated with expression of endogenous p55 IL-2 receptor chains. The role of the transfected p70 chains in formation of the high-affinity receptor sites was confirmed by the finding that the species-specific inhibitory antibody TU27 blocked high-affinity binding to human p70 and chimeric human/murine p70-expressing cells while having no effect on cells transfected with the murine p70 cDNA construct. As consequences of the expression of the transfected p70 chains, the cells responded to IL-2 with increased levels of endogenous p55 receptor subunit and both short and long-term proliferation. These studies substantiate the role of the p70 receptor chain in functional responses to IL-2 and provide a model system for future dissection of the structure/function relationships of the IL-2 receptor.

Detection of clonally expanded salivary gland lymphocytes in Sjögren's syndrome.

Recurrent swelling of the parotid and submandibular salivary glands occurs in some patients with Sjögren's syndrome (SS). Using Southern blot methods, we analyzed immunoglobulin and T cell antigen receptor (TCAR) gene rearrangements in DNA obtained from salivary gland lymphocytes of 9 SS patients. Based on histologic appearance of the biopsy specimens, these patients were diagnosed as having myoepithelial sialadenitis. We found oligoclonal rearrangements of the kappa gene (4 patients) and lambda gene (1 patient), and oligoclonal rearrangement of the TCAR beta chain in 2 additional SS patients. Patients with Ig gene rearrangements did not show rearrangements of their TCAR gene, nor did patients with TCAR rearrangements exhibit Ig rearrangements. The observed oligoclonal rearrangements probably derive from 5-10% of the salivary gland B cells or T cells. Three of these SS patients developed non-Hodgkin's lymphoma 2-8 years after the initial biopsy. Our results suggest that minor populations of B cells or T cells may clonally expand in the salivary gland tissues of SS patients with pseudolymphoma, and that such lymphocyte expansions may be controlled by the endogenous immune response and/or medications. However, continued lymphoproliferation in these salivary gland tissues may eventually lead to emergence of a neoplastic clone that escapes immunologic control and develops into a non-Hodgkin's lymphoma as a result of a multistep process.

Comparison of T cell receptor gene rearrangements in patients with large granular T cell leukemia and Felty's syndrome.

Felty's syndrome (FS) refers to the occurrence of rheumatoid arthritis, splenomegaly, and neutropenia. A subset of these patients has recently been described with a chronic T cell leukemia of large granular lymphocytes (LGCL). To examine the spectrum of lymphocyte abnormalities in FS and LGCL, we examined phenotypic and genotypic properties of lymphocytes from eight FS patients. In two of these FS patients, we observed an elevated proportion of T cells with an unusual phenotype (CD3+/Leu-7+/Leu-8-/CR3+) (46 +/- 5% of mononuclear cells). The FS lymphocytes had large granular morphology on Wright-Giemsa stain and were active in antibody-dependent cellular cytotoxic activity. This phenotype, morphology, and activity was similar to LGCL patients except that the latter T cells additionally expressed the Fc-IgG receptor recognized by monoclonal antibody Leu-11 (CD 15). In the remaining six FS patients, the proportion of CD3+/Leu-7+/CR 3+ T cells was only 10 +/- 8%, which was not significantly different from age-matched normal subjects (6.6 +/- 2.2%). To determine the clonality of T lymphocytes in FS and LGCL, we examined DNA for rearrangements of the T cell antigen receptor beta-chain (Ti beta) and gamma-chain (Ti gamma) genes by using Southern blotting techniques. We found a clonal rearrangement of the Ti beta 1 and Ti gamma genes in both LGCL patients. In contrast, no clonal rearrangements of Ti beta or Ti gamma genes were detected in lymphocytes from the FS patients. These results indicate that FS patients are heterogeneous in their phenotype and that one subset exhibits polyclonal expansion of an unusual lymphocyte subset.