Mycobacterium smegmatis expressing a chimeric protein MPT64-proteolipid protein (PLP) 139-151 reorganizes the PLP-specific T cell repertoire favoring a CD8-mediated response and induces a relapsing experimental autoimmune encephalomyelitis.

We infected SJL mice with a recombinant Mycobacterium smegmatis expressing a chimeric protein containing the self-epitope of proteolipid protein 139-151 (p139) fused to MPT64, a secreted protein of Mycobacterium tuberculosis (rMS(p139)). Infected mice developed a relapsing experimental autoimmune encephalomyelitis (EAE), showing a prevailing demyelination of the CNS, and disease severity was significantly lower in comparison with the one that follows immunization with p139. rMS(p139) was not detected in lymph node or spleen in the course of clinical disease development or in the CNS during relapse. Infection with rMS(p139) modified the p139-specific T cell repertoire, recruiting the spontaneous p139-specific repertoire and activating CD4(+) T cells carrying the BV4 semiprivate rearrangement. T cells carrying the public BV10 rearrangement that are consistently found in the CNS during flares of disease were not activated by infection with rMS(p139) because lymph node APCs infected with rMS(p139) selectively fail to present the epitope for which BV10 cells are specific. Simultaneously, rMS(p139) expanded p139-specific CD8(+) cells more efficiently than immunization with peptide in adjuvant. SJL mice vaccinated against the CDR3 sequence of the BV10 public rearrangement reduced usage of the BV10 cells and displayed reduced symptoms during bouts of EAE. Thus, transient peripheral infection with a CNS-cross-reactive nonpathogenic Mycobacterium induces a relapsing EAE that continues long after clearance of the infectious agent. The composition of the self-reactive repertoire activated determines severity and histology of the resulting disease.

Lipid incorporation inhibits Src-dependent assembly of fibronectin and type I collagen by vascular smooth muscle cells.

A vital role of vascular smooth muscle cells (SMCs) is to stabilize the artery wall by elaborating fibrils of type I collagen. This is especially important in atherosclerotic lesions. However, SMCs in these lesions can be laden with lipids and the impact of this modification on collagen fibril formation is unknown. To address this, we converted human vascular SMCs to a foam cell state by incubating them with either LDL or VLDL. Biochemical markers of a SMC phenotype were preserved. However, microscopic tracking revealed a profound perturbation in the ability of the cells to assemble collagen fibrils, reducing assembly by up to 79%. This dysfunction was mirrored by an inability of smooth muscle foam cells to assemble fibronectin. Lipid-loaded SMCs did not display a generalized defect in the actin cytoskeleton and the formation of vinculin-containing focal adhesion complexes was preserved. However, lipid-loaded SMCs were unable to assemble fibrillar adhesion complexes and clustering of tensin and alpha5beta1 integrin was disordered. Moreover, phosphorylation of tensin, required for fibrillar adhesion complex formation, was suppressed by up to 57%, with a concomitant decrease in activation of Src and FAK and restriction of activated Src to the cell edges. Forced activation of Src-FAK signaling in lipid-engorged SMCs rescued both fibrillar adhesion formation and fibrillogenesis. We conclude that lipid accumulation by SMCs disables the machinery for collagen and fibronectin assembly. This previously unknown relationship between atherogenic lipids and integrin-based signaling could underlie plaque vulnerability.

Src and FAK mediate cell-matrix adhesion-dependent activation of Met during transformation of breast epithelial cells.

Cell-matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand-independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non-neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto-activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with beta1 integrins and is co-localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src-dependent phosphorylation of Met requires cell-matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src-induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF-dependent and Src/integrin-dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells.

Disruption by interferon-alpha of an autocrine interleukin-6 growth loop in IL-6-dependent U266 myeloma cells by homologous and heterologous down-regulation of the IL-6 receptor alpha- and beta-chains.

IL-6 is an autocrine growth factor for U266 myeloma cells and their growth is inhibited by IFN-alpha or IL-6 mAb. We asked, therefore, whether IFN-alpha-induced growth inhibition involved IL-6. IFN-alpha and mAb against IL-6, the IL-6R alpha-(gp80) or beta-chain (gp130) potently inhibited U266 cells. Remarkably, this effect occurred despite IFN-alpha-augmented secretion of endogenous IL-6. However, examining the IL-6R revealed that IFN-alpha drastically curtailed expression of the IL-6R alpha- and beta-chain. This effect occurred on two different levels (protein and mRNA) and by two different mechanisms (directly and indirectly through IL-6). First, IFN-alpha, but not IL-6, greatly decreased gp80 and, to a lesser extent, gp130 mRNA levels which resulted in a loss of IL-6 binding sites. Second, IFN-alpha-induced IL-6 predominantly down-regulated membrane-bound gp130. IFN-alpha-mediated decrease of gp80 levels was not detected on IL-6-independent myeloma (RPMI 8226) or myeloid cells (U937). We conclude that IFN-alpha inhibited IL-6-dependent myeloma cell growth by depriving U266 cells of an essential component of their autocrine growth loop, a functional IL-6R.

Bryostatin-1 and IFN-gamma synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages.

Bryostatin-1 (Bryo) is a nontumor-promoting protein kinase C modulator that has been shown to have both in vitro and in vivo activity against several murine and human tumors. In this study, we investigated the effects of Bryo on nitric oxide production, measured as accumulated nitrite (NO2-) in culture supernatant, and inducible nitric oxide synthase (iNOS) gene expression in the murine macrophage cell line ANA-1. ANA-1 macrophages did not produce NO2- or iNOS mRNA constitutively, and very little or no NO2- or iNOS mRNA were detectable upon exposure to IFN-gamma. Bryo, although ineffective alone, and IFN-gamma synergized to produce high levels of NO2- and iNOS mRNA. The activity of Bryo was evident at a concentration of 0.1 ng/ml and reached its maximum at 1 ng/ml. The effects of Bryo were time dependent because expression of iNOS mRNA was detectable as early as 6 h and increased through 24 h. Analyses of the molecular mechanisms involved indicate that Bryo and IFN-gamma mainly regulate iNOS gene expression posttranscriptionally through stabilization of iNOS mRNA. Experiments designed to investigate the role of tumor necrosis factor alpha (TNF-alpha) in NO2- production by Bryo- and IFN-gamma-activated macrophages revealed that ANA-1 macrophages expressed low levels of TNF-alpha mRNA constitutively that were not augmented in the presence of IFN-gamma. However, Bryo alone augmented the TNF-alpha mRNA expression, which was only slightly increased with the addition of IFN-gamma. A polyclonal antibody to TNF-alpha was able to completely neutralize TNF-alpha secreted in either medium or Bryo plus IFN-gamma-treated cultures. Neutralizing concentrations of anti-TNF-alpha antibody suppressed the Bryo plus IFN-gamma-induced NO2- production approximately by 50%, suggesting that NO2- produced by Bryo plus IFN-gamma-treated ANA-1 macrophages may involve both TNF-alpha-dependent and TNF-alpha-independent mechanisms. Overall, these findings provide the first evidence that Bryo and IFN-gamma can synergize for the induction of NO2- production as well as iNOS gene expression and show the involvement of posttranscriptional mechanisms in the induction of iNOS mRNA.

Interleukin-2 and human monocyte activation.

The recognition of the monocyte/macrophage-activating properties of IL-2 has broadened our image of the biological effects of this lymphokine from those of a T cell growth factor to those of a molecule with pleiotropic effects. The detailed analysis of the mechanisms of action of IL-2 including its biological effects on different cell types and the regulation of its receptors has increased dramatically the spectrum of the biological responses that can be modified by IL-2. The regulation of the expression of the IL-2 receptor subunits differs in terms of response to extracellular stimuli and intracellular control, suggesting that the response to IL-2 will vary depending on the nature and extent of environmental stimulation. Furthermore, the fact that the IL-2R gamma chain can be part of the receptor for IL-4, IL-7, and perhaps other cytokines indicates that IL-2 may modulate the response of monocytes simply by binding or releasing the IL-2R gamma chain and thus modulating the responsiveness to IL-4 or IL-7. Conversely, the extent of utilization of IL-2R gamma chain by various cytokines may dictate the monocytic response to IL-2. In fact, the availability of IL-2R gamma chain seems to be the limiting factor in the response of monocytes to IL-2. Modulation of cytokine receptors is an integral part of the control of the IL-2 response. The induction of CSF-1 receptor by IL-2 and the positive effect of CSF-1 on the duration of the cytotoxic response in IL-2-stimulated monocytes are an interesting example of a synergistic interaction of potential physiological relevance. The response of monocytes to IL-2 can also be modulated by inhibitory circuits, such as those involving TGF-beta 1, IFN-gamma, and IL-4. However, IFN-gamma and IL-4 can also activate monocytes and the timing and relative concentrations of the various cytokines may be critical variables in determining the ultimate monocyte phenotype. These studies have given us a glimpse of a very complex picture composed of multiple backgrounds and several players. However, the present information is not sufficient to make meaningful predictions of the resulting monocyte phenotype in an inflammatory reaction in which multiple cytokines are involved.(ABSTRACT TRUNCATED AT 400 WORDS)

LPS-inducible nuclear factor in human monocytes that binds the negative regulatory element of the HIV LTR.

We studied the constitutive and lipopolysaccharide (LPS)-induced expression of nuclear protein binding to the negative regulatory element (NRE) of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in fresh human monocytes. We demonstrated the existence of a constitutive factor binding to the NRE 73-bp HpaII/HpaII fragment (-216 to -143) whose expression is up-regulated by LPS treatment. Competition experiments with overlapping oligonucleotides covering the HpaII/HpaII fragment and with mutated oligonucleotides mapped the binding within the TTTCATCAC region (-171 to -163). This binding pattern is unique to human monocytes.

Integrin VLA-6 (α6ß1) is transiently expressed during the development of mouse bone marrow-derived mast cells.

In the present study the involvement of VLA-6 (α6ß1) integrin, a laminin receptor, was characterized during the course of mouse bone marrow-derived mast cell (BMMC) development. Flow cytometry and immunoprecipitation revealed increases in α6 integrin expression during the first 3 weeks, followed by a decline, such that α6ß1 was no longer detectable by week 13. Using RT-PCR, transcripts for α6A but not the α6B isoform were detected. Results from immunoprecipitation and costaining with ß1-or ß4-specific mAb showed the expression of VLA-6 (α6ß1) and not α6ß4 heterodimers. Moreover, the ability of BMMC to interact with laminin correlated with the time period of VLA-6 expression. However, only 40% of adhesion to laminin was inhibited by blocking mAb for α6, indicating the involvement of additional laminin receptor(s). This is supported by the immunoprecipitation of VLA-2 integrin, also known to have laminin binding properties. Heterogeneity of VLA-6 expression was also found in connective tissue-type mast cells; thus, VLA-6hi and VLA-6lo subpopulations of peritoneal mast cells were observed. The heterogeneity of VLA-6 integrin expression in BMMC and CTMC may be relevant to the concept of mast cell heterogeneity as well as to the ability of mast cell precursors to migrate and complete their course of maturation within tissues.

IL-6 signals inhibition of cell adhesion in melanoma A375-C6.

IL-6 has been found to be a potent inhibitor of melanoma A375-C6 cell adhesion, in addition to its known action in arresting cells at G1/G0 phase of the cell cycle IL-6 treated melanoma cells were found to round up and to lose the ability to adhere to fibronectin, laminin, collagen, and tenascin over 72 to 96 hours of IL-6 treatment, a time course similar to that seen for cell cycle inhibition. Cell cycle inhibition and loss of adhesion were found, however, to be independent effects of IL-6. Analysis of cell surface integrins indicated significant changes in the expression of several integrins including downregulation of a3 and av beta 5 and upregulation of a3. However, the changes in integrin expression did not correlate with loss of adhesion to relevant ligands. Three A375 melanoma clones varying in metastatic potential also demonstrated inhibition of both cell proliferation and matrix adhesion by IL-6.

Perilymphatic injections of cytokines: a new tool in active cancer immunotherapy. Experimental rationale and clinical findings.

Various strategies for local immunotherapy performed by using lymphokines and lymphocytes are presented. In experimental mice models, the antitumor reaction elicited through the injection of little amounts of IL-2 and gamma-IFN at the tumor growth site is significantly enhanced by the concurrence of lymphocytes from tumor bearing mice artificially admixed to challenging tumor cells. These lymphocytes act as initiator or helper cells that elicit both a local and a systemic long lasting immune reaction by the release of additional lymphokines. The reaction activated mostly involves draining lymph nodes, where multiple reaction mechanisms are induced. The local injection of cocktails of molecularly defined lymphokines can also mimic the helper action of lymphocytes and trigger a similarly effective anti-tumor reaction. A set of pilot clinical trials with IL-2 or gamma-IFN injected perilymphatically were initiated in patients with advanced primary or recurrent localized tumors of the head and neck and the bladder. This treatment appears to elicit a local reaction by activating a few pathways of patients immunoreactivity, whose efficiency may well be high, as shown by the tumor inhibition often observed.

Coordinated integrin and growth factor regulation of primary keratinocyte migration mediated through extracellular signal regulated kinase and phosphoinositide 3-kinase.

We have examined coordinated integrin and growth factor regulation of primary keratinocyte migration mediated by phosphoinositide 3-kinase (PI3K) and mitogen-activated extracellular-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK). On collagen I and fibronectin substrates, both epidermal growth factor (EGF) and hepatocyte growth factor (HGF) stimulated chemokinetic (random) and chemotactic (directional) migration. On provisional matrix, a combination of fibronectin and fibrin found in the early phase of wound healing, EGF and HGF-stimulated significant chemotactic but little or no chemokinetic cell movement. Blocking mAbs to integrin alpha2beta1 and alpha5beta1 effectively inhibited EGF- and HGF-stimulated chemokinetic and chemotactic cell movement on collagen I and fibronectin, respectively; however, HGF-stimulated chemotactic migration on collagen I was only partially inhibited by alpha2beta1 blocking mAb. Differentiated keratinocytes underwent reduced chemokinetic and chemotactic migration compared with undifferentiated keratinocytes; however, EGF-stimulated migration was reduced more than HGF-stimulated migration. When the migratory response on collagen I and fibronectin was assessed in the presence of the MEK-specific inhibitor PD98059, EGF- and HGF-stimulated chemotaxis was significantly reduced, whereas PD98059 had little effect on the stimulated chemokinesis. PI3K-specific inhibitor LY294002 reduced EGF- and HGF-stimulated chemokinesis and chemotaxis on collagen I and fibronectin. Thus beta1 integrins acted in concert with EGF and HGF to regulate migration of primary keratinocytes on extracellular matrix components via PI3K and MEK/ERK.

The extracellular domain of CD11d regulates its cell surface expression.

A mAb targeting the CD11d subunit of the leukocyte integrin CD11d/CD18 decreases intraspinal inflammation and oxidative damage leading to improved neurological outcomes in rodent models of SCI. CD11d/CD18 is the fourth member of the beta2-integrin family. Current evidence indicates that CD11d/CD18 is regulated differently than other beta2-integrins, suggesting that CD11d(+) leukocytes play a distinct role in inflammation. Although the transcriptional control of CD11d expression has been evaluated, control of the intracellular distribution of CD11d has not been addressed. For this reason and as a result of the potential of CD11d as a therapeutic target for SCI and possibly other CNS injuries, we investigated the intracellular localization and surface expression of CD11d in cultured cells. CD11d and CD18 were fused at their C-termini with YFP and mRFP, respectively. Flow cytometry and confocal microscopy demonstrated that rCD11d-YFP is expressed on the cell surface of leukocyte cell lines expressing CD18. In contrast, in heterologous cell lines, CD11d-YFP is retained intracellularly in the TGN. Coexpression of CD11d-YFP and CD18-mRFP relieves this intracellular restriction and allows the CD11d/CD18 heterodimer to be surface-expressed. Based on domain-swapping experiments with CD25, the extracellular domain of CD11d is required and sufficient for the observed intracellular retention in heterologous cells. Furthermore, the transmembrane and C-terminus are also required for proper heterodimerization with CD18 and localization to the plasma membrane. These findings suggest that multiple CD11d domains play a role in controlling intracellular location and association with CD18.

Cathepsin B is involved in the trafficking of TNF-alpha-containing vesicles to the plasma membrane in macrophages.

TNF-alpha is a potent proinflammatory cytokine, essential for initiating innate immune responses against invading microbes and a key mediator involved in the pathogenesis of acute and chronic inflammatory diseases. To identify molecules involved in the production of TNF-alpha, we used a functional gene identification method using retroviral integration-mediated mutagenesis, followed by LPS-stimulated TNF-alpha production analysis in macrophages. We found that cathepsin B, a lysosomal cysteine proteinase, was required for optimal posttranslational processing of TNF-alpha in response to the bacterial cell wall component LPS. Mouse bone marrow-derived macrophages from cathepsin B-deficient mice and macrophages treated with the cathepsin B-specific chemical inhibitor CA074 methyl ester or small interfering RNA against cathepsin B secreted significantly less TNF-alpha than wild-type or nontreated macrophages. We further showed that the inhibition of cathepsin B caused accumulation of 26-kDa pro-TNF-containing vesicles. Ectopic expression of GFP-conjugated pro-TNF further suggests that pro-TNF failed to reach the plasma membrane without intracellular cathepsin B activity. Altogether, these data suggest that intracellular cathepsin B activity is involved in the TNF-alpha-containing vesicle trafficking to the plasma membrane.

Interaction of epidermal growth factor, Ca2+, and matrix metalloproteinase-9 in primary keratinocyte migration.

Elevations of epidermal growth factor (EGF) and Ca(2+) concentrations in the wound site are associated with reepithelialization during wound healing. In addition, Ca(2+) and EGF can both induce increases in matrix metalloproteinase-9 (MMP-9) synthesis. However, little is known about the interplay of these events in regulating the migration properties of primary keratinocytes on collagen I, the most abundant extracellular matrix component in the skin. We found that EGF stimulated both chemokinetic and chemotactic migration of primary keratinocytes on collagen I; however, MMP-9 was required for EGF-stimulated chemotaxis but not EGF-stimulated chemokinesis. Calcium at 0.5 mM stimulated chemokinetic migration of keratinocytes. Together, Ca(2+) and EGF stimulated higher levels of chemokinesis than either stimulus alone. Furthermore, Ca(2+) could restore the ability of keratinocytes from MMP-9 null mice to undergo EGF-stimulated chemotaxis. The phosphatidylinositol-3 kinase inhibitor LY294002 inhibited both EGF- and Ca(2+)-stimulated chemokinetic migration. In contrast, the MEK inhibitor PD98059 blocked Ca(2+)- but not EGF-stimulated chemokinetic migration of keratinocytes. A combination of PD98059 and LY294002 was required to inhibit Ca(2+) enhancement of EGF-stimulated migration completely. Calcium-stimulated chemokinesis was completely blocked by either the protein kinase C-alpha inhibitor Gö6976 or the src/fyn inhibitor PP2. Using primary keratinocytes, our results showed how the combined action of Ca(2+), EGF, and MMP-9 regulated the contributions of extracellular-regulated kinase and phosphatidylinositol-3 kinase toward chemokinetic and chemotactic migration of keratinocytes.

Chemokine receptor CXCR4-beta1 integrin axis mediates tumorigenesis of osteosarcoma HOS cells.

It is known that beta1 integrins mediate the migratory response of cells to chemokine stimulation. Also, both beta1 integrins and chemokines have roles in tumor development. In the present study, the beta1 integrin-chemokine axis is assessed using human osteosarcoma (HOS) transfectant cells expressing the CXCR4 receptor for chemokine SDF-1 (CXCL12). We first identified in vitro the specific beta1 integrins that mediated the migratory response to SDF-1 stimulation. Results showed that on collagen type I and laminin, the chemotactic response to SDF-1 was predominantly mediated by alpha2beta1 integrin. On fibronectin, SDF-1-stimulated chemotaxis involved both alpha4beta1 and alpha5beta1 integrins. A comparison of the transfectant clones expressing CXCR4 at low, intermediate, and high levels and the control transfectant revealed that the transfectant clones migratory response in vitro and their ability to form tumors in vivo was related to their levels of CXCR4 expression. In addition, treatment by injection with mAbs to CXCR4, integrin alpha2beta1, or integrin alpha5beta1 effectively inhibited the growth of HOS-CXCR4 transfectant cells in vivo. Therefore, our results show that the beta1 integrins that mediated the migratory response were also functionally linked to the enhanced tumor growth of CXCR4-expressing HOS transfectant cells.

Ability of interleukin-4 to elicit an immunoreaction against a murine tumor.

A T cell-derived lymphokine, IL-4, has been shown to have activating and growth promoting properties on macrophages, T cells and B cells. IL-4 could thus be used to augment immunoreactivity in the treatment of tumors. To evaluate its ability to promote an immune reaction capable of hampering tumor growth, we injected mice with small amounts of recombinant IL-4 perilymphatically daily for ten days near the challenge site of a poorly immunogenic syngenic fibrosarcoma (CE-2). These low doses activated such a strong immune reaction that incipient syngenic tumors were rejected. Our results suggest that the host immune system plays a fundamental role in this IL-4 dependent lymphokine-activated tumor inhibition (LATI).

Picolinic acid- or desferrioxamine-inducible autocrine activation of macrophages engineered to produce IFNgamma: an approach for gene therapy.

Macrophage (Mphi)-based vectors are highly mobile cellular shuttles designed to deliver therapeutic genes within the tissues. We engineered a mouse Mphi cell line to express the murine interferon-gamma (IFNgamma) under the control of an inducible promoter containing the hypoxia-responsive element, which can be triggered by hypoxia and other stimuli. We show that this Mphi vector can be induced to produce IFNgamma under normoxic conditions by stimulation with picolinic acid (PA), a catabolite of tryptophan, or desferrioxamine (DFX), an iron-chelating drug. The Mphi vector responds to IFNgamma with the induction of IRF-1 and of other IFNgamma-inducible genes, the expression of Ia antigens and induction of phagocytic activity. Inducible nitric oxygen synthase gene expression, nitric oxide production, as well as TNFalpha secretion were enhanced by PA or DFX as the sole stimuli. None of the above responses could be triggered individually by PA or DFX in control, normal Mphi, indicating that the Mphi vector overcame the need for costimulatory molecules derived from the immune system for its full activation. Furthermore, we demonstrate that extracellular iron can downregulate such response, thereby identifying an additional tool for the fine tuning of the Mphi vector response to stimulation.