Lena Axelsson

Leukocyte Activation Detected by Increased Plasma Levels of Inflammatory Mediators in Patients With Ischemic Cerebrovascular Diseases

BACKGROUND AND PURPOSE Leukocytes have been implicated in the development of ischemic atherosclerotic vascular diseases. In a prospective study we investigated whether the plasma concentrations of inflammatory mediators, ie, proteases and cytokines, as markers for systemic leukocyte activation, are increased in patients with acute ischemic cerebrovascular diseases. METHODS Using enzyme-linked immunosorbent assays, we measured the plasma levels of neutrophil gelatinase-associated lipocalin (NGAL), neutrophil proteinase 4 (NP4), tumor necrosis factor-alpha (TNF), and soluble TNF receptor protein-1 p55 (sTNFR-1) in 120 patients with acute ischemic cerebrovascular insult (72 with stroke and 48 with transient ischemic attack [TIA]) and in 35 age- and sex-matched healthy subjects. RESULTS Compared with the control group, plasma NGAL levels were higher in the stroke group (P < .0001) and the TIA group (P < .01); plasma NP4 levels were higher in the stroke group (P < .0001) and the TIA group (P < .01); and plasma sTNFR-1 levels were higher in the stroke group (P < .04). There was significant correlation between the plasma levels of fibrinogen and those of both sTNFR-1 (r = .32; P = .005) and NGAL (r = .40; P = .0001) and between the erythrocyte sedimentation rate and the plasma levels of both sTNFR-1 (r = .35; P = .001) and NGAL (r = .34; P = .002). CONCLUSIONS Our study demonstrated that markers for systemic leukocyte activation, ie, plasma levels of cytokines and proteases, were higher in patients with acute ischemic cerebrovascular disease than in healthy control subjects. Activated leukocytes and leukocytic mediators may have an important role in acute cerebrovascular ischemia and its consequences.

Studies of the release and turnover of a human neutrophil lipocalin

A 24-kDa protein was purified from human neutrophil extracts and shown to be the newly discovered neutrophil gelatinase-associated lipocalin (NGAL), based on structural and immunochemical data. A specific enzyme-linked immunosorbent assay (ELISA) was developed for the determination of NGAL in human plasma and tissue fluids. Normal human plasma contains 72 micrograms l-1 of NGAL (range 40-109 micrograms l-1) in two main forms, monomer and dimer. 35S-methionine metabolic studies of human neutrophils showed that granulocyte macrophagecolony-stimulating factor (GMCSF) stimulated significant synthesis and secretion of NGAL in a dose- and time-dependent fashion. NGAL was rapidly released as monomer and dimer on incubation of heparinized whole blood with opsonized yeast, reaching a plateau corresponding to about 35% of total cell content after 30 min. Following intravenous injection of 125-iodine labelled NGAL there was a more rapid initial clearance of the monomeric than of the dimeric form; t1/2 10 min vs. 20 min. During the second phase the two forms cleared at similar rates. Severe acute peritonitis was accompanied by a 10-fold increase in NGAL plasma levels and the NGAL level in peritoneal exudates, which reached about 40 mg l-1. There was a good linear correlation between the concentrations of NGAL, leucocyte elastase and NP4 (neutrophil proteinase 4 = P3).

A t-butyloxycarbonyl-modified Wnt5a-derived hexapeptide functions as a potent antagonist of Wnt5a-dependent melanoma cell invasion

The influential role of Wnt5a in tumor progression underscores the requirement for developing molecules that can target Wnt5a-mediated cellular responses. In the aggressive skin cancer, melanoma, elevated Wnt5a expression promotes cell motility and drives metastasis. Two approaches can be used to counteract these effects: inhibition of Wnt5a expression or direct blockade of Wnt5a signaling. We have investigated both options in the melanoma cell lines, A2058 and HTB63. Both express Frizzled-5, which has been implicated as the receptor for Wnt5a in melanoma cells. However, only the HTB63 cell line expresses and secretes Wnt5a. In these cells, the cytokine, TGFβ1, controlled the expression of Wnt5a, but due to the unpredictable effects of TGFβ1 signaling on melanoma cell motility, targeting Wnt5a signaling via TGFβ1 was an unsuitable strategy to pursue. We therefore attempted to target Wnt5a signaling directly. Exogenous Wnt5a stimulation of A2058 cells increased adhesion, migration and invasion, all crucial components of tumor metastasis, and the Wnt5a-derived N-butyloxycarbonyl hexapeptide (Met-Asp-Gly-Cys-Glu-Leu; 0.766 kDa) termed Box5, abolished these responses. Box5 also inhibited the basal migration and invasion of Wnt5a-expressing HTB63 melanoma cells. Box5 antagonized the effects of Wnt5a on melanoma cell migration and invasion by directly inhibiting Wnt5a-induced protein kinase C and Ca2+ signaling, the latter of which we directly demonstrate to be essential for cell invasion. The Box5 peptide directly inhibits Wnt5a signaling, representing an approach to anti-metastatic therapy for otherwise rapidly progressive melanoma, and for other Wnt5a-stimulated invasive cancers.

Release of neutrophil proteinase 4(3) and leukocyte elastase during phagocytosis and their interaction with proteinase inhibitors

Neutrophil proteinase 4 (NP4) is a major neutral proteinase of the human polymorphonuclear (PMN) leukocyte, which is present in amounts similar to leukocyte elastase. NP4(3) is a potent, non-specific proteinase, which may degrade structural and soluble proteins in the tissues and body fluids, and it has been implicated as an important pathogenetic factor in lung emphysema. We have studied the release of elastase and NP4(3) in an in vitro model of phagocytosis. alpha 1-proteinase inhibitor (alpha 1-PI) is the major plasma inhibitor of both leukocyte elastase and NP4(3), but alpha 1-PI bound leukocyte elastase more readily than NP4(3). The basic conditions were designed so that some proteolytic activity was present in the medium. Addition of increasing amounts of Secretory leukocyte protease inhibitor (SLPI) to the incubation mixtures resulted in binding of leukocyte elastase to this inhibitor and extinction of free proteolytic activity against both natural and synthetic substrates. The progressive binding of leukocyte elastase to SLPI instead of alpha 1-PI was paralleled by an increasing binding of NP4(3) to alpha 1-PI. SLPI is a potent inhibitor of leukocyte elastase and cathepsin G, and although it lacks inhibitory effect on NP4(3), it may obviously indirectly aid in the binding and inhibition of NP4(3) to alpha 1-PI, by taking care of at least part of the leukocyte elastase. As a specific NP4(3)-inhibitor is not readily available for therapeutic use, this effect may prove useful under in vivo conditions and enhance the protective effect of administered recombinant human SLPI.

Down-regulation of Rac activity during beta 2 integrin-mediated adhesion of human neutropolis.

In human neutrophils, β2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the β2 integrin-induced down-regulation of Rac activities. In contrast, β2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the β2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The β2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by β2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a β2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.

Release of cytokines and proteases from human peripheral blood mononuclear and polymorphonuclear cells following phagocytosis and LPS stimulation

Release and cellular contents of pro- and anti-inflammatory cytokines, neutrophilic elastase and secretory leukocyte proteinase inhibitor (SLPI) were measured with enzyme-linked immunosorbent assay in peripheral blood mono- and polymorphonuclear cells stimulated with preopsonized yeast cells or lipopolysaccharide. Tumour necrosis factor alpha (TNF alpha) was also measured with a bioassay. TNF alpha production and soluble TNF alpha receptor I (sTNF RI) were demonstrated in the environment of both cell populations. The bioassay indicated levels of TNF alpha far below those detected by ELISA. The overall secretion of cytokines and their inhibitors was found to favour an anti-inflammatory balance in the environment of the stimulated cells. The interleukin-1 receptor antagonist (IL1-ra), compared with interleukin-1 beta (IL-1 beta), dominated the secretions from both cell types with a 100- to 1000-fold excess respectively. Most of the translated IL-1 beta was not secreted but found associated with the cellular compartments. In contrast to lipopolysaccharide (LPS) stimulation, preopsonized yeast cells stimulated a massive release of elastase from neutrophil cells.

Nitric Oxide Produced in Response to Engagement of beta 2 Integrins on Human Neutrophils Activates the Monomeric GTPases Rap1 and Rap2 and Promotes Adhesion

We found that engagement of β2 integrins on human neutrophils increased the levels of GTP-bound Rap1 and Rap2. Also, the activation of Rap1 was blocked by PP1, SU6656, LY294002, GF109203X, or BAPTA-AM, which indicates that the downstream signaling events in Rap1 activation involve Src tyrosine kinases, phosphoinositide 3-kinase, protein kinase C, and release of calcium. Surprisingly, the β2 integrin-induced activation of Rap2 was not regulated by any of the signaling pathways mentioned above. However, we identified nitric oxide as the signaling molecule involved in β2 integrin-induced activation of Rap1 and Rap2. This was illustrated by the fact that engagement of β2 integrins increased the production of nitrite, a stable end-product of nitric oxide. Furthermore, pretreatment of neutrophils with Nω-monomethyl-l-arginine, or 1400W, which are inhibitors of inducible nitric-oxide synthase, blocked β2 integrin-induced activation of Rap1 and Rap2. Similarly, Rp-8pCPT-cGMPS, an inhibitor of cGMP-dependent serine/threonine kinases, also blunted the β2 integrin-induced activation of Rap GTPases. Also nitric oxide production and its downstream activation of cGMP-dependent serine/threonine kinases were essential for proper neutrophil adhesion by β2 integrins. Thus, we made the novel findings that β2 integrin engagement on human neutrophils triggers production of nitric oxide and its downstream signaling is essential for activation of Rap GTPases and neutrophil adhesion.

Interleukin-6 drives melanoma cell motility through p38α-MAPK-dependent up-regulation of WNT5A expression

Extensive research has demonstrated a tumor‐promoting role of increased WNT5A expression in malignant melanoma. However, very little light has been shed upon how WNT5A expression is up‐regulated in melanoma. A potential regulator of WNT5A expression is the pro‐inflammatory cytokine Interleukin (IL)‐6, which shares the ability of WNT5A to increase melanoma cell invasion. Here, we investigate whether IL‐6 can promote melanoma cell motility through an increased expression of WNT5A. We clearly demonstrate that the WNT5A‐antagonistic peptide Box5 could inhibit IL‐6‐induced melanoma cell migration and invasion. Furthermore, IL‐6 stimulation of the human melanoma cell lines HTB63 and A375 increased the expression of WNT5A in a dose‐dependent manner. To identify the signaling mechanism responsible for this up‐regulation, we explored the involvement of the three main signals induced by IL‐6; STAT3, Akt and ERK 1/2. Of these, only STAT3 was activated by IL‐6 in the melanoma cell lines tested. However, the STAT3 inhibitor S3I‐201 failed to inhibit IL‐6‐induced WNT5A up‐regulation in HTB63 and A375 cells. Nor did STAT3 siRNA silencing affect the expression of WNT5A. In search of an alternative signaling mechanism, we detected IL‐6‐induced activation of p38‐MAPK in HTB63 and A375 cells. The p38‐MAPK inhibitor SB203580 abolished the IL‐6‐induced WNT5A up‐regulation and blocked IL‐6‐induced melanoma cell invasion. The latter effect could be rescued by the addition of recombinant WNT5A. Notably, immunoprecipitation analysis revealed that only the p38α‐MAPK isoform was activated by IL‐6, and subsequent siRNA silencing of p38α‐MAPK abolished the IL‐6‐induced up‐regulation of WNT5A. Taken together, we demonstrate a novel link between the two melanoma pro‐metastatic agents IL‐6 and WNT5A explaining how IL‐6 can increase melanoma cell invasion and thus promote the metastatic process. This finding provides a basis for future therapeutic intervention of melanoma progression.

WNT-5A triggers Cdc42 activation leading to an ERK1/2 dependent decrease in MMP9 activity and invasive migration of breast cancer cells.

An important role for WNT‐5A is implicated in a variety of tumors, including breast carcinoma. We previously showed that WNT‐5A signaling inhibits migration and metastasis of breast cancer cells, and that patients with primary breast cancer in which WNT‐5A was expressed have a better prognosis. Despite the fact that RhoGTPase Cdc42 is commonly associated with increased cell migration, we here show that recombinant WNT‐5A activates the Cdc42 in breast cancer cells (lines MDA‐MB468 and MDA‐MB231) in a time‐dependent manner. Activation of Cdc42 was also observed in MDA‐MB468 cells that were stably transfected with a WNT‐5A plasmid (MDA‐MB468‐5A). In all situations, increased Cdc42 activity was accompanied by decreased migration and invasion of the breast cancer cells. To explore these findings further we also investigated the effect of WNT‐5A signaling on ERK1/2 activity. Apart from an initial Ca2+‐dependent rWNT‐5A‐induced activation of ERK1/2, Cdc42 activity was inversely correlated with ERK1/2 activity in both rWNT‐5A‐stimulated parental MDA‐MB468 and MDA‐MB468‐5A cells. We also demonstrated increased ERK1/2 activity in MDA‐MB468‐5A cells following siRNA knockdown of Cdc42. Consistent with these results, breast cancer cells transfected with constitutively active Cdc42 exhibited reduced ERK1/2 activity, migration and invasion, whereas cells transfected with dominant negative Cdc42 had increased ERK1/2 activity in response to rWNT‐5A. To gain information on how ERK1/2 can mediate its effect on breast cancer cell migration and invasion, we next investigated and demonstrated that WNT‐5A signaling and constitutively active Cdc42 both decreased matrix metalloproteinase 9 (MMP9) activity. These data indicate an essential role of Cdc42 and ERK1/2 signaling and MMP9 activity in WNT‐5A‐impaired breast cancer cells.

Migration and invasion of oral squamous carcinoma cells is promoted by WNT5A, a regulator of cancer progression

BACKGROUND Oral squamous cell carcinoma (OSCC) constitutes 90% of all cancers in the oral cavity, and the prognosis for patients diagnosed with OSCC is still poor. The identification of novel therapeutic targets and prognostic markers for OSCC is therefore essential. Previous studies of OSCC revealed an increased expression of WNT5A in the tumor tissue. However, no functional studies of WNT5A-induced effects in OSCC have been performed. METHODS Two different OSCC cell lines were used for analysis of WNT5A expression by Western blot, whereas WNT5A-induced responses were analyzed by measuring calcium (Ca²⁺) signaling, PKC activation, migration and invasion. RESULTS Despite the lack of WNT5A expression, both cell lines responded to recombinant WNT5A (rWNT5A) with activation of the non-canonical WNT/Ca²⁺ /PKC pathway. This effect was ascertained to be mediated by WNT5A by use of the WNT5A antagonist, Box5. To investigate how WNT5A affects tumor progression, rWNT5A-induced alterations in BrdU absorbance (reflecting the number of tumor cells) were analyzed. rWNT5A had no effect on BrdU absorbance but instead promoted tumor cell migration and invasion. These results were confirmed by the use of the WNT5A-mimicking peptide Foxy5, while the rWNT5A-induced migration was blocked by secreted Frizzled-related protein 1 (SFRP1), protein kinase C inhibitors or the intracellular Ca²⁺ chelator, MAPT. CONCLUSIONS These novel data clearly show that WNT5A activates the non-canonical WNT/Ca²⁺ /PKC pathway and increases migration and invasion of OSCC cells. This may indicate how an increased WNT5A expression in the tumor tissue is likely to promote progression of OSCC.