Matrix metalloproteinases: they're not just for matrix anymore!

The matrix metalloproteinases (MMPs) have been viewed as bulldozers, destroying the extracellular matrix to permit normal remodeling and contribute to pathological tissue destruction and tumor cell invasion. More recently, the identification of specific matrix and non-matrix substrates for MMPs and the elucidation of the biological consequence of cleavage indicates that perhaps MMPs should be viewed more as pruning shears, playing sophisticated roles in modulating normal cellular behavior, cell-cell communication and tumor progression.

Tumor progression: defining the soil round the tumor seed.

The tumor microenvironment, or stroma, is known to contribute to tumor progression. Two recent studies have shown that the stromal protein matrix metalloproteinase MMP-9 has a role in the early stages of tumor growth and angiogenesis.

Elevation of intracellular cAMP inhibits growth factor-mediated matrix metalloproteinase-9 induction and keratinocyte migration.

Receptor tyrosine kinases are regulators of diverse cellular functions including cell growth, cell survival, differentiation, locomotion, and morphogenesis. Activation of the cAMP-dependent protein kinase A inhibits receptor tyrosine kinase-stimulated growth responses in a number of cell types. In this study, we investigated the consequences of elevated cAMP on growth factor-mediated keratinocyte migration and matrix metalloproteinase (MMP)-9 induction in a human keratinocyte cell line. We found that elevation of intracellular cAMP by forskolin abolishes epidermal growth factor (EGF)- or scatter factor/hepatocyte growth factor-dependent colony dispersion. Concentrations of forskolin that inhibit growth factor-induced motility also eliminate EGF- or scatter factor/hepatocyte growth factor-dependent induction of the 92-kDa gelatinase/MMP-9. In contrast to findings obtained in fibroblasts, elevated intracellular cAMP did not interfere with growth factor-dependent activation of the p42/44 extracellular signal-regulated kinases, indicating that cAMP-dependent inhibition of migration and MMP-9 induction does not occur through perturbation of the extracellular signal-regulated kinases/mitogen-activated protein kinase pathway. However, forskolin effectively inhibited EGF-dependent activation of c-Jun N-terminal kinase and p38, demonstrating that cAMP selectively interferes with a different subset of growth factor-induced mitogen-activated protein kinase signaling cascades than reported previously in fibroblasts. These findings illustrate that EGF concurrently activates multiple mitogen-activated protein kinase signaling cascades in keratinocytes and suggests that each pathway contributes to maximal EGF-dependent migration and proteinase induction.

Matrix metalloproteinases: multifunctional contributors to tumor progression.

Matrix metalloproteinases (MMPs) are a family of extracellular matrix degrading proteinases. Owing to their matrix-degrading abilities and high expression in advanced tumors, MMPs were originally implicated in invasion and metastasis during cancer progression. However, recent work extends a role for MMPs during multiple stages of tumor progression to include other functions such as growth, angiogenesis and migration. Based on studies in animal models implicating MMP activity in cancer, synthetic MMP inhibitors are currently being tested in a clinical setting.

Sustained activation of the mitogen-activated protein kinase pathway. A mechanism underlying receptor tyrosine kinase specificity for matrix metalloproteinase-9 induction and cell migration.

Activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway is required for ligand-dependent regulation of numerous cellular functions by receptor tyrosine kinases. We have shown previously that although many receptor tyrosine kinase ligands are mitogens for keratinocytes, cell migration and induction of the 92-kilodalton gelatinase/matrix metalloproteinase (MMP)-9 are selectively regulated by the epidermal growth factor and scatter factor/hepatocyte growth factor receptors. In this report we present evidence of an underlying mechanism to account for these observed differences in receptor tyrosine kinase-mediated response. Ligands that are mitogenic, but do not induce MMP-9 or colony dispersion, transiently activate the p42/p44 ERK/MAP kinases. In contrast, ligands that stimulate MMP-9 induction and colony dispersion induced sustained activation of these kinases. The functional significance of sustained MAPK activation was demonstrated by inhibition of the MAP kinase kinase MEK1. Disruption of the prolonged signal by addition of the MEK1 inhibitor PD 98059 up to 4 h after growth factor stimulation substantially impaired ligand-dependent colony dispersion and MMP-9 induction. These findings support the conclusion that duration of MAPK activation is an important determinant for certain growth factor-mediated functions in keratinocytes.

Contributions of the epidermal growth factor receptor to keratinocyte motility.

The epidermal growth factor (EGF) receptor plays a central role in numerous aspects of keratinocyte biology. In normal epidermis, the EGF receptor is important for autocrine growth of this renewing tissue, suppression of terminal differentiation, promotion of cell survival, and regulation of cell migration during epidermal morphogenesis and wound healing. In wounded skin, the EGF receptor is transiently up-regulated and is an important contributor to the proliferative and migratory aspects of wound reepithelialization. In keratinocytic carcinomas, aberrant expression or activation of the EGF receptor is common and has been proposed to play a role in tumor progression. Many cellular processes such as altered cell adhesion, expression of matrix degrading proteinases, and cell migration are common to keratinocytes during wound healing and in metastatic tumors. The EGF receptor is able to regulate each of these cellular functions and we propose that transient and dynamic elevation of EGF receptor during wound healing, or constitutive overexpression in tumors, provides an important contribution to the migratory and invasive potential of keratinocytes.

Epidermal growth factor (EGF)- and scatter factor/hepatocyte growth factor (SF/HGF)- mediated keratinocyte migration is coincident with induction of matrix metalloproteinase (MMP)-9.

Receptor tyrosine kinases are key regulators of cellular function including cell growth, differentiation, migration, and morphogenesis. Disruptions of receptor tyrosine kinase signaling pathways are often associated with changes in cellular proliferative capacity and tumorigenesis. Both receptor-specific and cell type-specific factors may contribute to the ultimate cellular responses observed after receptor activation. In this regard, we find that both normal keratinocytes and their tumorigenic counterparts display differential responses to activation of receptor tyrosine kinases. Multiple ligands were mitogenic for keratinocytes, but only epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), and scatter factor/hepatocyte growth factor (SF/HGF) promoted cell motility as assessed by colony dispersion (scattering) and in vitro reepithelialization. Interestingly, growth factor specificity for motility coincided with ligand-mediated cell invasion through a reconstituted basement membrane and induction of the 92-kDa metalloproteinase (MMP-9) activity as determined by gelatin zymogram analysis. Inhibitors of MMP activity or addition of an MMP-9 neutralizing antibody resulted in the loss of growth factor-induced colony dispersion, suggesting a functional role for MMP-9 induction during this response. Coordinate regulation of MMP-9 induction and the migratory response are likely to contribute to the enhanced invasive potential observed in response to EGF and SF/HGF. Our findings suggest that alternate receptor-mediated signaling pathways leading to differences in gene expression may be involved in complex cellular responses such as colony dispersion or invasion.

Overexpression of the epidermal growth factor receptor contributes to enhanced ligand-mediated motility in keratinocyte cell lines.

In keratinocytes, epidermal growth factor (EGF) promotes cell motility in addition to proliferation. As EGF receptor expression is elevated during wound healing and in many epithelial tumors, we wanted to investigate whether there is a direct relationship between EGF receptor expression and ligand-mediated cellular locomotion. EGF receptor activation induced cell migration in normal keratinocytes and their tumorigenic counterparts; however, the rate of colony dispersion and in vitro reepithelialization was more rapid in the squamous cell carcinoma (SCC) lines that exhibited elevated (> or = 5-fold) EGF receptor levels. Within a single SCC line, submaximal concentrations of EGF or reduction of EGF receptor activity by an anti-EGF receptor neutralizing antibody resulted in delayed kinetics of in vitro reepithelialization. Thus, suppression of EGF receptor activity in an overexpressing SCC line restores a migratory response that more closely resembles that of normal keratinocytes. Conversely, ligand-induced colony dispersion was augmented in stable clonal cell lines in which EGF receptor expression was elevated after introduction of an EGF receptor complementary DNA construct. Collectively, these findings suggest that the migratory potential of keratinocytes is modulated at the level of both receptor expression and ligand concentration, with a positive correlation between EGF receptor levels and ligand-induced cell motility.

Cytokine elevations in infants with bacterial and aseptic meningitis.

We sought to determine whether the detection of cytokines, produced during the inflammatory response, would aid in the diagnosis of meningitis in young infants. We measured cerebrospinal fluid (CSF) and plasma levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF) in 62 infants less than 6 months of age whose condition was evaluated for meningitis. Twenty infants had culture-proved meningitis, 22 had aseptic meningitis, and 20 control infants had no evidence of meningitis. The CSF IL-6 levels were elevated in all 20 infants with bacterial meningitis and in 9 of 22 infants with aseptic meningitis but were undetectable in all control subjects. Furthermore, CSF IL-6 levels were 10 times greater in infants with bacterial versus aseptic meningitis (p < 0.001). Levels of TNF in CSF were detected in 12 of 20 infants with bacterial meningitis and were undetectable in infants with aseptic meningitis and in control infants (p < 0.02). Plasma IL-6 and TNF levels were unreliable for the detection of meningitis in this patient population. We conclude that the presence of IL-6 in the CSF reliably identifies infants with meningitis and that the presence of CSF TNF is a highly specific indicator of bacterial meningeal inflammation.

Cromolyn inhibits assembly of the NADPH oxidase and superoxide anion generation by human neutrophils.

Early and late phase reactions have been observed in asthma; the late phase reaction is characterized by accumulation of inflammatory cells such as neutrophils. Activated neutrophils degranulate and assemble an active NADPH oxidase, which generates superoxide anion (O2-), reactions that have been implicated in lung tissue damage. Preincubation of neutrophils with the asthma drug cromolyn sodium selectively inhibited FMLP (10(-7) M) and PMA (0.1 microgram/ml) elicited O2- generation but not degranulation. To further characterize the mechanism of this inhibition we examined the effect of cromolyn on the NADPH oxidase complex and the signaling pathways for its assembly. Ca2+ mobilization and activation of protein kinase C have been implicated as signals for activation of the NADPH oxidase. Ca2+ mobilization triggered by FMLP was significantly decreased by 21.2% in cromolyn-treated cells. In contrast, cromolyn did not interfere with translocation or activity of protein kinase C. Membranes prepared from neutrophils stimulated with 0.5 microgram/ml PMA generated O2-, indicating assembly of an active NADPH oxidase; cromolyn did not inhibit this membrane-associated, preassembled oxidase. In contrast, preincubation of neutrophils with 100 microM cromolyn before addition of PMA decreased the capacity of the membranes to generate O2- by 57.3%. These results indicate that cromolyn inhibited the assembly of an active NADPH oxidase. The efficacy of cromolyn may be associated with inhibition of assembly of an active NADPH oxidase in the neutrophil and prevention of oxygen radical-induced tissue damage.

In vitro and in vivo effects of granulocyte colony-stimulating factor on neutrophils in glycogen storage disease type 1B: granulocyte colony-stimulating factor therapy corrects the neutropenia and the defects in respiratory burst activity and Ca2+ mobilization.

Children with glycogen storage disease (GSD) type 1b are susceptible to recurrent bacterial infections and have chronic neutropenia accompanied by phagocytic cell dysfunction including decreased superoxide anion (O2-) generation, calcium (Ca2+) mobilization, and chemotactic activity. Granulocyte colony-stimulating factor (G-CSF), a cytokine that corrects neutropenia in other diseases, in vitro enhances f-Met-Leu-Phe-triggered neutrophil O2- generation. Short-term pretreatment (15 min) of GSD 1b neutrophils with G-CSF increased the rate of O2- production (p < 0.01); however, this rate was still significantly below the rate of O2- production in control neutrophils. Recombinant human G-CSF (5 micrograms/kg/d) was administered s.c. to a GSD 1b patient. Before treatment, absolute neutrophil counts were < 500/mm3. Two d after G-CSF administration, the absolute neutrophil counts increased to 1333 and remained in the normal range during a 12-mo follow-up period. In vivo, G-CSF therapy increased f-Met-Leu-Phe-stimulated O2- production to 52% of control after 1 mo, and by mo 4, O2- production reached control levels. Our previous studies (J Clin Invest 56:196-202, 1990) demonstrated that decreased O2- production in neutrophils was associated with impaired Ca2+ mobilization. In vivo administration of G-CSF increased f-Met-Leu-Phe-triggered Ca2+ mobilization by neutrophils to 43% of control by mo 1 of G-CSF therapy and to 93% of control by mo 4, thus paralleling the improvements in O2- generation. In contrast, G-CSF therapy had no effect on the defective neutrophil chemotaxis. In summary, G-CSF therapy produced a rapid increase in circulating neutrophils and a gradual correction of O2- production.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokine elevations in critically ill infants with sepsis and necrotizing enterocolitis.

We hypothesized that plasma levels of cytokines such as interleukin-6 and tumor necrosis factor (TNF) are elevated in critically ill infants with sepsis and necrotizing enterocolitis (NEC) and that the magnitude of their elevation is correlated with mortality rate. We measured plasma levels of interleukin-6 and TNF in 62 newborn infants with suspected sepsis or NEC. Eighteen infants had bacterial sepsis, 9 had bacterial sepsis plus NEC, and 15 had NEC but negative culture results. Twenty comparably ill infants with negative results on culture of systemic specimens served as study control subjects. Interleukin-6 levels were five- to tenfold higher in infants with bacterial sepsis plus NEC at the onset of disease than in infants with bacterial sepsis alone, in infants with NEC but negative culture results, and in control infants (p < 0.01). These differences persisted throughout the 48-hour study period. Interleukin-6 levels were also significantly higher in nonsurvivors than in survivors (p < 0.001). In contrast, plasma TNF values were not consistently increased in any of the groups. We conclude that plasma interleukin-6 is a more reliable indicator of bacterial sepsis and NEC than plasma TNF and may identify infants who might benefit from immunotherapeutic strategies.

Interferon-gamma corrects the respiratory burst defect in vitro in monocyte-derived macrophages from glycogen storage disease type 1b patients.

Glycogen storage disease (GSD) type 1b is accompanied by decreased respiratory burst activity in peripheral blood phagocytic cells (i.e. monocytes and neutrophils). To elucidate whether this depressed respiratory burst was due to an intrinsic defect of phagocytic cells or due in part to in vivo host factors, we examined superoxide anion (O2-) production in monocytes from five GSD 1b patients cultured 9 d in vitro to allow for differentiation into macrophages (MDM). O2- production in MDM was measured in response to concanavalin A, fMet-Leu-Phe, and phorbol myristate acetate (PMA) stimulation. GSD 1b MDM had significantly depressed O2- generation with fMet-Leu-Phe and concanavalin A stimulation; however, unlike peripheral blood monocytes, GSD 1b MDM responded to PMA stimulation with O2- production comparable to healthy control donors. The cytokine interferon-gamma (IFN-gamma) has been shown to enhance O2- production in MDM. When GSD 1b MDM were cultured in the presence of IFN-gamma (1 x 10(5) U/L), O2- production in response to fMet-Leu-Phe, concanavalin A, and PMA was enhanced to rates similar to those of control MDM cultured in the presence of IFN-gamma. Thus, the respiratory burst defect observed in circulating phagocytic cells is also present in vitro in cultured GSD 1b MDM. However, in contrast to circulating phagocytic cells, depressed O2- production in GSD 1b MDM is selective to receptor-mediated activation, but not to PMA stimulation. This defect is correctable after short-term treatment with IFN-gamma, suggesting a role for IFN-gamma in treating the phagocytic defect in this disease.

Alpha-1-antichymotrypsin inhibits the NADPH oxidase-enzyme complex in phorbol ester-stimulated neutrophil membranes.

The generation of superoxide anion and release of granule contents are essential to the bactericidal function of neutrophils, but may also contribute to host tissue damage during inflammation. In previous studies (J. Immunol. 146:2388), we have demonstrated that the acute phase reactant alpha-1-antichymotrypsin (ACT), a potent inhibitor of the serine protease cathepsin G, also suppresses superoxide anion generation. The inhibitory effect of ACT was not directly linked to its antiproteolytic activity and may reflect interaction at a site other than its reactive loop. To further characterize the mechanism of inhibition, we investigated the direct effects of ACT on the NADPH oxidase enzyme complex and the signaling pathways that regulate motivation of the respiratory burst. We present evidence that ACT does not intefer with agonist-stimulated calcium mobilization or translocation and activity of protein kinase C. ACT was an effective inhibitor of superoxide anion generation in membrane preparations isolated from PMA-activated cells. These results support the notion that ACT is acting on a component of the active assembled NADPH oxidase complex. Thus, ACT may have an important role in regulation of specific aspects of the inflammatory processes and the modulation of toxic oxygen-based host tissue damage.

Isolation and flow cytometric characterization of newborn mouse brain-derived microglia maintained in vitro.

Microglia have been identified in the white matter of developing and adult mouse brain using different murine macrophage markers. While several techniques for the isolation of murine microglia have been described, the small cell yields and partial purification have limited the progress of these studies. We now describe the isolation of murine microglia using a modification of McCarthy and de Vellis method. Brain tissues from 1-2 day old newborn mice were mechanically and chemically dissociated and maintained in in vitro culture for 3 weeks. In primary dissociated brain cultures, microglia are observed after 10 days migrating from small colonies. After 16-20 days, brain-derived microglia were isolated with high cell yields by continuous shaking of the cultures for 16 hr. In contrast to resident murine peritoneal macrophages, microglia express less Class II (Ia) antigen and a small percentage express L3T4 (CD4) antigen by flow cytometry.

Isolation and characterization of newborn rabbit brain-derived microglia.

We isolated brain microglia from newborn rabbits and maintained these cells in in vitro culture. Enriched populations of rabbit microglia share several characteristics of mononuclear phagocytes including intracellular staining for nonspecific esterase and acid phosphatase. Microglia express Fc receptors, generate superoxide anion, and stain positive with the lectin Ricinus communis. Rabbit brain microglia develop multinucleated giant cells and small colonies in in vitro culture. The cells are highly phagocytic in culture. Other investigators have recently demonstrated that rabbits can be infected with HIV-1 in vivo and that neurological symptoms occur only when HIV-1 infection was carried out in HTLV-1-infected rabbits. Brain microglia most likely play a central role in HIV-1 encephalopathy. The availability of rabbit brain microglia in in vitro culture, offers a valuable potential cell model to study HIV-1 infection in the central nervous system.