Targeting NF-kappaB in Waldenstrom macroglobulinemia

The nuclear factor-B (NF-B) path-way has been implicated in tumor B-cell survival, growth, and resistance to therapy. Because tumor cells overcome single-agent antitumor activity, we hypothesized that combination of agents that target differentially NF-B pathway will induce significant cytotoxicity. Therapeutic agents that target proteasome and Akt pathways should induce significant activity in B-cell malignancies as both pathways impact NF-B activity. We demonstrated that perifosine and bortezomib both targeted NF-B through its recruitment to the promoter of its target gene IB using chromatin immunoprecipitation assay. This combination led to synergistic cytotoxicity in Waldenstrom macroglobulinemia (WM) cells that was mediated through a combined reduction of the PI3K/Akt and ERK signaling pathways, found to be critical for survival of WM cells. Moreover, a combination of these drugs with the CD20 monoclonal antibody rituximab further increased their cytotoxic activity. Thus, effective WM therapy may require combination regimens targeting the NF-B pathway.      

Regulation of tumor necrosis factor receptor-1 and the IKK-NF-kappaB pathway by LDL receptor-related protein explains the antiinflammatory activity of this receptor

Low-density lipoprotein receptor–related protein (LRP-1) functions in endocytosis and in cell signaling directly (by binding signaling adaptor proteins) or indirectly (by regulating levels of other cell-surface receptors). Because recent studies in rodents suggest that LRP-1 inhibits inflammation, we conducted activity-based protein profiling experiments to discover novel proteases, involved in inflammation, that are regulated by LRP-1. We found that activated complement proteases accumulate at increased levels when LRP-1 is absent. Although LRP-1 functions as an endocytic receptor for C1r and C1s, complement protease mRNA expression was increased in LRP-1–deficient cells, as was expression of inducible nitric oxide synthase (iNOS) and interleukin-6. Regulation of expression of inflammatory mediators was explained by the ability of LRP-1 to suppress basal cell signaling through the IB kinase–nuclear factor-B (NF-B) pathway. LRP-1–deficient macrophages, isolated from mice, demonstrated increased expression of iNOS, C1r, and monocyte chemoattractant protein-1 (MCP-1); MCP-1 expression was inhibited by NF-B antagonism. The mechanism by which LRP-1 inhibits NF-B activity involves down-regulating cell-surface tumor necrosis factor receptor-1 (TNFR1) and thus, inhibition of autocrine TNFR1-initiated cell signaling. TNF-–neutralizing antibody inhibited NF-B activity selectively in LRP-1–deficient cells. We propose that LRP-1 suppresses expression of inflammatory mediators indirectly, by regulating TNFR1-dependent cell signaling through the IB kinase–NF-B pathway.      

The NF-kappaB subunit Rel A is associated with in vitro survival and clinical disease progression in chronic lymphocytic leukemia and represents a promising therapeutic target

In this study, we characterized nuclear factor B (NF-B) subunit DNA binding in chronic lymphocytic leukemia (CLL) samples and demonstrated heterogeneity in basal and inducible NF-B. However, all cases showed higher basal NF-B than normal B cells. Subunit analysis revealed DNA binding of p50, Rel A, and c-Rel in primary CLL cells, and Rel A DNA binding was associated with in vitro survival (P = .01) with high white cell count (P = .01) and shorter lymphocyte doubling time (P = .01). NF-B induction after in vitro stimulation with anti-IgM was associated with increased in vitro survival (P < .001) and expression of the signaling molecule ZAP-70 (P = .003). Prompted by these data, we evaluated the novel parthenolide analog, LC-1, in 54 CLL patient samples. LC-1 induced apoptosis in all the samples tested with a mean LD₅₀ of 2.8 µM after 24 hours; normal B and T cells were significantly more resistant to its apoptotic effects (P < .001). Apoptosis was preceded by a marked loss of NF-B DNA binding and sensitivity to LC-1 correlated with basal Rel A DNA binding (P = .03, r² = 0.15). Furthermore, Rel A DNA binding was inversely correlated with sensitivity to fludarabine (P = .001, r² = 0.3), implicating Rel A in fludarabine resistance. Taken together, these data indicate that Rel A represents an excellent therapeutic target for this incurable disease.     

CD4+ CD25+ Treg cells inhibit human memory gammadelta T cells to produce IFN-gamma in response to M tuberculosis antigen ESAT-6

T cells play an important role in innate immunity against infections; however, the regulation of these cells remains largely unknown. In the present study, we show that ESAT-6, an antigen of Mycobacterium tuberculosis, induces IFN- secretion by human T cells. In addition, ESAT-6 also induces the activation and proliferation of T cells. Phenotypic analysis indicates that IFN-–producing T cells are mainly effector memory cells with the surface phenotype of CD45RA^–CD62L^–CCR7^–. These results were further confirmed by the fact that naive T cells from cord blood did not produce IFN- in response to ESAT-6. Further studies indicated that stimulation with ESAT-6 directly induced purified T cells to produce IFN-, independent of both antigen-presenting cells and CD4⁺ T cells. Unexpectedly, depletion of CD4⁺ T cells markedly enhanced IFN- production by T cells, indicating that CD4⁺ T cells regulate the response of T cells. Importantly, CD4⁺CD25⁺ T regulatory (Treg) cells but not CD4⁺CD25^– T cells significantly inhibited IFN- production by T cells. Taken together, these data demonstrate for the first time that Treg cells can play an important role in the regulation of immune responses of antigen-specific human memory T cells.     

Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells

Several cell-based immunotherapy strategies have been developed to specifically modulate T cell–mediated immune responses. These methods frequently rely on the utilization of tolerogenic cell–based antigen-presenting cells (APCs). However, APCs are highly sensitive to cytotoxic T-cell responses, thus limiting their therapeutic capacity. Here, we describe a novel bead-based approach to modulate T-cell responses in an antigen-specific fashion. We have generated killer artificial APCs (aAPCs) by coupling an apoptosis-inducing -Fas (CD95) IgM mAb together with HLA-A₂ Ig molecules onto beads. These aAPCs deplete targeted antigen-specific T cells in a Fas/Fas ligand (FasL)–dependent fashion. T-cell depletion in cocultures is rapidly initiated (30 minutes), dependent on the amount of aAPCs and independent of activation-induced cell death (AICD). aAPCs represent a novel technology that can control T cell–mediated immune responses, and therefore has potential for use in treatment of autoimmune diseases and allograft rejection.     

A functional 14-3-3zeta-independent association of PI3-kinase with glycoprotein Ib alpha, the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex

Engagement of the adhesion receptor glycoprotein (GP) Ib-IX-V by von Willebrand factor (VWF) mediates platelet adhesion to damaged vessels and triggers platelet activation and thrombus formation in heart attack and stroke. GPIb-IX-V contains distinct 14-3-3–binding sites at the GPIb C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)–dependent site on GPIbβ involving Ser166. 14-3-3 regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3 association blocks receptor signaling, suggesting a key functional role for 14-3-3. We used deletion mutants of GPIb expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3 binding to another GPIb-IX-V–associated signaling protein, phosphoinositide 3-kinase (PI3-kinase). Pull-down experiments involving glutathione S-transferase (GST)–PI3-kinase/p85-subunit and GST–14-3-3 indicated that both proteins interacted with contiguous GPIb sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIb expressed in CHO cells, inhibited VWF/ristocetin-dependent Akt phosphorylation, relative to wild-type receptor, confirming this region encompassed a functional PI3-kinase–binding site. Pull-down experiments with GST-p85 truncates indicated the GPIb-binding region involved the p85 breakpoint cluster region (BCR) domain, containing RSXSXP. However, pull-down of GPIb-IX was unaltered by mutation/deletion/phosphorylation of this potential 14-3-3–binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIb independently of 14-3-3; 14-3-3 inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3 but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3. Together, these data suggest the GPIb C-terminus regulates signaling through independent association of 14-3-3 and PI3-kinase.     

Interruption of the NF-kappaB pathway by Bay 11-7082 promotes UCN-01-mediated mitochondrial dysfunction and apoptosis in human multiple myeloma cells

Interactions between pharmacologic NF-B inhibitors (eg, Bay 11-7082, SN-50) and the checkpoint abrogator UCN-01 have been examined in human multiple myeloma (MM) cells. Exposure of U266 cells to Bay 11-7082 (Bay) in combination with UCN-01 resulted in the abrogation of NF-B/DNA binding activity and the synergistic induction of apoptosis. Comparable synergism was observed in other MM cell lines and patient-derived CD138⁺ cells and between an inhibitory peptide of NF-B (SN50) and UCN-01. Bay/UCN-01-mediated lethality involved mitochondrial dysfunction, caspase cleavage, and poly adenosine diphosphate-ribose polymerase (PARP) degradation. Although Bay modestly blocked UCN-01-induced extracellular signal-regulated kinase (ERK) phosphorylation, coadministration activated c-Jun N-terminal kinase (JNK) and cdc2/cdk1 and down-regulated Mcl-1, XIAP, and Bcl-x_L. Transfection with a constitutively activated mitogen-activated protein kinase kinase (MEK1)/green fluorescent protein (GFP) construct failed to block apoptosis induced by Bay/UCN-01 but significantly attenuated MEK inhibitor (U0126)/UCN-01-induced lethality. Inhibiting JNK activation with SP600125 or D-JNKI1 peptide markedly reduced Bay/UCN-01-mediated mitochondrial dysfunction and apoptosis and the down-regulation of Mcl-1, XIAP, and Bcl-x_L but not of cdc2/cdk1 activation. Stable transfection of cells with dominant-negative caspase-9 dramatically diminished Bay/UCN-01 lethality without altering JNK or cdc2/cdk1 activation. Neither interleukin-6 (IL-6)- nor fibronectin-mediated adherence conferred resistance to Bay/UCN-01-induced apoptosis. Together, these findings suggest that a strategy combining UCN-01 with disruption of the IB kinase (IKK)/IB/NF-B pathway warrants attention in MM. (Blood. 2004;103:2761-2770)      

Direct interaction between Kit and the interleukin-7 receptor

In vivo analyses of thymopoiesis in mice defective in signaling through Kit and c or Kit and IL-7R demonstrate synergy and partial complementation of c or IL-7–mediated signaling by the Kit signaling pathway. Our molecular analysis in T-lymphoid cells as well as in nonhematopoietic cells shows that Kit and IL-7R signaling pathways directly interact. KL-mediated activation of Kit induced strong tyrosine phosphorylation of c and IL-7R in the absence of IL-7. Activated Kit formed a complex with either IL-7R or c, and tyrosine phosphorylation of both subunits occurred independently of Jak3, suggesting that c and IL-7R are each direct substrates of Kit. Kit activated Jak3 in an IL-7R–dependent manner. Moreover, deficient Stat5 activation of the Kit mutant YY567/569FF lacking intrinsic Src activation capacity was partially reconstituted in the presence of IL-7R and Jak3. Based on the molecular data, we propose a model of Kit-mediated functional activation of c-containing receptors such as IL-7R, similar to the interaction between Kit and Epo-R. Such indirect activation of the Jak-Stat pathway induced by the interaction between an RTK and type I cytokine receptor could be the underlying mechanism for a context-specific signaling repertoire of a pleiotropic RTK-like Kit.     

Induction of the IL-9 gene by HTLV-I Tax stimulates the spontaneous proliferation of primary adult T-cell leukemia cells by a paracrine mechanism

The etiologic agent of adult T-cell leukemia (ATL) is human T cell lymphotropic virus type I (HTLV-I). The HTLV-I protein Tax alters gene expression, including those of cytokines and their receptors, which plays an important role in early stages of ATL. Here we demonstrate that expression of interleukin-9 (IL-9) is activated by Tax via an NF-B motif in its proximal promoter, whereas IL-9 receptor- (IL-9R) expression is not induced by Tax. However, supporting a role for IL-9/IL-9R in ATL, a neutralizing monoclonal antibody directed toward IL-9R inhibited ex vivo spontaneous proliferation of primary ATL cells from several patients. Fluorescence-activated cell sorter analysis of freshly isolated peripheral blood mononuclear cells from these patients revealed high level expression of IL-9R on their CD14-expressing monocytes. Furthermore, purified T cells or monocytes alone from these patients did not proliferate ex vivo, whereas mixtures of these cell types manifested significant proliferation through a contact-dependent manner. Taken together, our data suggest that primary ATL cells, via IL-9, support the action of IL-9R/CD14-expressing monocytes, which subsequently support the ex vivo spontaneous proliferation of malignant T cells. In summary, these data support a role for IL-9 and its receptor in ATL by a paracrine mechanism.      

Cytokine storm in a mouse model of IgG-mediated hemolytic transfusion reactions

Cytokines are hypothesized to play a central role in the pathophysiology of IgG-mediated hemolytic transfusion reactions (HTRs), and deeper understanding is required for improving therapy for these events. After establishing well-defined mouse models of HTRs, we tested whether cytokines were involved. Red blood cells (RBCs) from human glycophorin A transgenic (hGPA-Tg) or wild-type (WT) mice were transfused into non-Tg recipients passively immunized with monoclonal antibodies (Mabs). Only transfusions of incompatible RBCs induced IgG-mediated HTRs, exemplified by rapid clearance and hemoglobinuria. Very high plasma levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6), and lower levels of tumor necrosis factor- (TNF-), were induced after incompatible transfusion. No significant changes in IL-10, IL-12, or interferon- (IFN-) levels were observed. The proinflammatory cytokines elaborated in this in vivo mouse model are also implicated in the systemic inflammatory response syndrome (SIRS) and confirm the hypothesis that cytokine storm occurs as a result of HTRs.     

Alpha4beta1 integrin and 190-kDa CD44v constitute a cell surface docking complex for gelatinase B/MMP-9 in chronic leukemic but not in normal B cells

As B-cell chronic lymphocytic leukemia (B-CLL) progresses, malignant cells extravasate and infiltrate lymphoid tissues. Several molecules, including gelatinase B/MMP-9, contribute to these processes. Although mainly a secreted protease, some MMP-9 is present at the B-CLL cell surface and the function, mode of anchoring, and interactions of this MMP-9 are unknown. Here we show that anti–MMP-9 antibodies immunoprecipitated a 190-kDa CD44v isoform and 4β1 integrin from B-CLL cells, but not from normal B cells. Function-blocking antibodies to 4β1 or CD44, or transfection with specific siRNAs, decreased cell-associated proMMP-9 and increased the secreted form. B-CLL cells attached to and bound proMMP-9 and active MMP-9, and this was inhibited by blocking the expression or function of 4β1 or CD44. The MMP-9 hemopexin domain was critical in these interactions. 4β1 and 190-kDa CD44v (but not CD44H) formed a complex at the cell surface, since they both coimmunoprecipitated with anti-4, anti-β1, or anti-CD44 antibodies. Immunofluorescence analyses confirmed that 4β1 and CD44v colocalized with MMP-9. Binding of proMMP-9 inhibited B-CLL cell migration, and this required MMP-9 proteolytic activity. Thus, we have identified 4β1 and CD44v as a novel proMMP-9 cell surface docking complex and show that cell-associated MMP-9 may regulate B-CLL cell migration and arrest.     

Restrictive usage of monoclonal immunoglobulin lambda light chain germline in POEMS syndrome

POEMS syndrome is a rare plasma cell disorder characterized by peripheral neuropathy, monoclonal gammopathy, and high levels of serum vascular endothelial growth factor, the pathogenesis of which remains unclear. A unique feature of this syndrome is that the proliferating monoclonal plasma cells are essentially -restricted. Here we determined complete nucleotide sequences of monoclonal immunoglobulin light chain (IGL) variable regions in 11 patients with POEMS syndrome. The V-region of the Ig gene of all 11 patients was restricted to the V1 subfamily. Searching for homologies with IGL germlines revealed that 2 germlines, IGLV1-44*01 (9/11) and IGLV1-40*01 (2/10), were identified, with an average homology of 91.1%. The IGLJ3*02 gene was used in 11 of 11 re-arrangements with an average homology of 92.2%. These data suggest that the highly restricted use of IGL V1 germlines plays an important role in the pathogenesis of POEMS syndrome.     

Integrin alpha E(CD103)beta 7 influences cellular shape and motility in a ligand-dependent fashion

While the extravasation cascade of lymphocytes is well characterized, data on their intraepithelial positioning and morphology are scant. However, the latter process is presumably crucial for many immune functions. Integrin _E(CD103)β₇ has previously been implicated in epithelial retention of some T cells through binding to E-cadherin. Our current data suggest that _E(CD103)β₇ also determines shape and motility of some lymphocytes. Time-lapse microscopy showed that wild-type _E(CD103)β₇ conferred the ability to form cell protrusions/filopodia and to move in an amoeboid fashion on E-cadherin, an activity that was abrogated by _E(CD103)β₇-directed antibodies or cytochalasin D. The _E-dependent motility was further increased (P < .001) when point-mutated _E(CD103) locked in a constitutively active conformation was expressed. Moreover, different yellow fluorescent protein–coupled _E(CD103) species demonstrated that the number and length of filopodia extended toward purified E-cadherin, cocultured keratinocytes, cryostat-cut skin sections, or epidermal sheets depended on functional _E(CD103). The in vivo relevance of these findings was demonstrated by wild-type dendritic epidermal T cells (DETCs), which showed significantly more dendrites and spanned larger epidermal areas as compared with DETCs of _E(CD103)-deficient mice (P < .001). Thus, integrin _E(CD103)β₇ is not only involved in epithelial retention, but also in shaping and proper intraepithelial morphogenesis of some leukocytes.     

Type I NKT cells protect (and type II NKT cells suppress) the host's innate antitumor immune response to a B-cell lymphoma

Natural killer T (NKT) cells are a T-cell subpopulation known to possess immunoregulatory functions and recognize CD1d molecules. The majority of NKT cells express an invariant T-cell receptor (TCR) chain rearrangement (V14J18 in mice; V24J18 in humans) and are called type I NKT cells; all other NKT cells are type II. In the current study, we have analyzed the roles for these NKT-cell subsets in the host's innate antitumor response against a murine B-cell lymphoma model in vivo. In tumor-bearing mice, we found that type I NKT cells conferred protection in a CD1d-dependent manner, whereas type II NKT cells exhibited inhibitory activity. Pro- and anti-inflammatory cytokines secreted by splenocytes from tumor-bearing mice correlated with tumor progression. Myeloid cells (CD11b⁺Gr1⁺) were present in large numbers at the tumor site and in the spleen of tumor-bearing type I NKT–deficient mice, suggesting that antitumor immunosurveillance was inhibited by CD11b⁺Gr1⁺ cells. Overall, these data suggest that there are distinct roles for NKT-cell subsets in response to a B-cell lymphoma in vivo, pointing to potential novel targets to be exploited in immunotherapeutic approaches against blood cancers.     

A310 helical turn is essential for the proliferation-inhibiting properties of macrophage inflammatory protein-1 alpha (CCL3)

Despite possessing marked structural similarities, the chemokines macrophage inflammatory protein-1 (MIP-1; CCL3) and RANTES (CCL5) display differential activity in hematopoietic progenitor-cell-inhibitory assays, with MIP-1 being active and RANTES inactive in this context. We have sought to identify the key structural determinants of this property of MIP-1. This has involved constructing MIP-1/RANTES chimeras by swapping structural domains between the 2 proteins. Results indicate that, in contrast to other chemokine functions, neither the N nor the C termini are key determinants of inhibitory activity. The motif that appears to be most important for this activity lies between the second and fourth cysteines of MIP-1 and further domain swap analysis has narrowed this down to the 3₁₀ helical turn preceding the first -strand in MIP-1. More detailed analysis has highlighted the role played by a specific dipeptide motif in the proliferation-inhibitory activity of chemokines. The involvement of the 3₁₀ helical-turn motif in chemokine function is unprecedented and this study therefore identifies a novel, functionally essential motif within chemokines. In addition, this study further attests to the alternative mechanisms of action used by MIP-1 in inhibition of hematopoietic progenitor-cell proliferation and regulation of leukocyte migration.      

Adult murine hematopoiesis can proceed without beta1 and beta7 integrins

The function of 41 and 47 integrins in hematopoiesis is controversial. While some experimental evidence suggests a crucial role for these integrins in retention and expansion of progenitor cells and lymphopoiesis, others report a less important role in hematopoiesis. Using mice with a deletion of the 1 and the 7 integrin genes restricted to the hematopoietic system we show here that 41 and 47 integrins are not essential for differentiation of lymphocytes or myelocytes. However, 17 mutant mice displayed a transient increase of colony-forming unit (CFU-C) progenitors in the bone marrow and, after phenylhydrazine-induced anemia, a decreased number of splenic erythroid colony-forming units in culture (CFUe's). Array gene expression analysis of CD4⁺CD8⁺ double-positive (DP) and CD4^–CD8^– double-negative (DN) thymocytes and CD19⁺ and CD4⁺ splenocytes did not provide any evidence for a compensatory mechanism explaining the mild phenotype. These data show that 41 and 47 are not required for blood cell differentiation, although in their absence alterations in numbers and distribution of progenitor cells were observed.      

Modulation of angiogenesis by omega-3 polyunsaturated fatty acids is mediated by cyclooxygenases

The potential role of dietary fats in cancer is attracting considerable interest within the community. Both epidemiologic and experimental findings suggest that omega-3 polyunsaturated fatty acids (-3 PUFAs), which are almost absent from typical Western diets, exert protective effects against cancer progression, although the precise mechanism of this suppression remains unknown. One of the potential targets for -3 PUFAs in cancer suppression is angiogenesis, a process of new blood vessel formation within rapidly growing tumors. Here, we demonstrate that -6 PUFAs stimulate and -3 PUFAs inhibit major proangiogenic processes in human endothelial cells, including the induction of angiopoietin-2 (Ang2) and matrix metalloprotease-9, endothelial invasion, and tube formation, that are usually activated by the major -6 PUFA arachidonic acid. The cyclooxygenase (COX)–mediated conversion of PUFAs to prostanoid derivatives participated in modulation of the expression of Ang2. Thus, the -6 PUFA–derived prostaglandin E2 augmented, whereas the -3 PUFA–derived prostaglandin E3 suppressed the induction of Ang2 by growth factors. Our findings are consistent with the suggestion that PUFAs undergo biotransformation by COX-2 to lipid mediators that modulate tumor angiogenesis, which provides new insight into the beneficial effects of -3 PUFAs.      

The Src family kinase Hck regulates mast cell activation by suppressing an inhibitory Src family kinase Lyn

IgE/antigen-dependent mast cell activation plays a central role in immediate hypersensitivity and other allergic reactions. The Src family tyrosine kinase (SFK) Lyn is activated by the cross-linking of high-affinity IgE receptors (FcRI). Activated Lyn phosphorylates the FcRI subunits, ß and , leading to subsequent activation of various signaling pathways. Lyn also plays a negative regulatory function by activating negative regulatory molecules. Another SFK, Fyn, also contributes to mast cell degranulation by inducing Gab2-dependent microtubule formation. Here we show that a third SFK, Hck, plays a critical role in mast cell activation. Degranulation and cytokine production are reduced in FcRI-stimulated hck^–/– mast cells. The reduced degranulation can be accounted for by defects in Gab2 phosphorylation and microtubule formation. Importantly, Lyn activity is elevated in hck^–/– cells, leading to increased phosphorylation of several negative regulators. However, positive regulatory events, such as activation of Syk, Btk, JNK, p38, Akt, and NF-B, are substantially reduced in hck^–/– mast cells. Analysis of lyn^–/–hck^–/–, lyn^–/–FcRIß^–/–, and hck^–/–FcRIß^–/– cells shows that Hck exerts these functions via both Lyn-dependent and Lyn-independent mechanisms. Thus, this study has revealed a hierarchical regulation among SFK members to fine-tune mast cell activation.      

Signalling, inflammation and arthritis: NF-kappaB and its relevance to arthritis and inflammation

In the synovial cells of patients with RA, activation of thenuclear factor-B (NF-B) pathway results in the transactivationof a multitude of responsive genes that contribute to the inflammatoryphenotype, including TNF- from macrophages, matrix metalloproteinasesfrom synovial fibroblasts and chemokines that recruit immunecells to the inflamed pannus. This is largely a consequenceof activation of the ‘canonical’ NF-B pathway thatinvolves heterodimers of p50/p65. Whilst much information onthe role of NF-B in inflammation has been gleaned from geneticdeficiency of the respective genes in mice, important differencesexist in the signalling networks between human and murine immunecells and immortalized cell lines. Despite these differencesat the molecular level, the importance of NF-B in inflammationis undisputed and inhibition of the pathway is widely believedto have great potential as a therapeutic target in RA. Commercialeffort has gone into developing inhibitors of NF-B activation.However, inhibition of the NF-B activation can result in anexacerbation of inflammation if TNF- production by macrophagesis not controlled. It will be important that such inhibitorsare carefully monitored before their long-term use in chronicinflammatory conditions such as RA. KEY WORDS: NF-B, Signalling pathways, Review Submitted 13 July 2007; revised version accepted 4 October 2007.     

Placenta growth factor augments endothelin-1 and endothelin-B receptor expression via hypoxia-inducible factor-1 alpha

Pulmonary hypertension (PHT) develops in sickle cell disease (SCD) and is associated with high mortality. We previously showed that erythroid cells produce placenta growth factor (PlGF), which activates monocytes to induce proinflammatory cytochemokines, contributing to the baseline inflammation and severity in SCD. In this study, we observed that PlGF increased expression of endothelin-1 (ET-1) and endothelin-B receptor (ET-BR) from human pulmonary microvascular endothelial cells (HPMVECs) and monocytes, respectively. PlGF-mediated ET-1 and ET-BR expression occurred via activation of PI-3 kinase, reactive oxygen species and hypoxia inducible factor-1 (HIF-1). PlGF increased binding of HIF-1 to the ET-1 and ET-BR promoters; this effect was abrogated with mutation of hypoxia response elements in the promoter regions and HIF-1 siRNA and confirmed by chromatin immunoprecipitation analysis. Furthermore, PlGF-mediated ET-1 release from HPMVECs and ET-BR expression in monocytes creates a PlGF–ET-1–ET-BR loop, leading to increased expression of MCP-1 and IL-8. Our studies show that PlGF-induced expression of the potent vasoconstrictor ET-1 and its cognate ET-BR receptor occur via activation of HIF-1, independent of hypoxia. PlGF levels are intrinsically elevated from the increased red cell turnover in SCD and in other chronic anemia (eg, thalassemia) and may contribute to inflammation and PHT seen in these diseases.