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Mark E Mummert

Publications and source records attributed to Mark E Mummert.

10 recordsLinked to original sources
PubMed2006

Tumor microenvironment modulates hyaluronan expression: the lactate effect.

Hyaluronan (HA) synthesis is a tightly regulated process and is partly controlled by the microenvironment (e.g., lactate concentration). Experimental evidence has indicated that the melanoma cells that synthesize large amounts of HA exhibit enhanced tumor cell growth and increased metastatic capacity compared to those expressing smaller amounts. Because most studies have examined HA expression on melanoma cells in vitro, we compared the patterns of HA expression by B16-F1 and B16-F10 melanoma cells in vitro and in situ. Cell surface HA expression was assessed with the HA-binding peptide Pep-1. B16-F1 melanoma cells showed significantly higher levels of Pep-1 binding compared with B16-F10 cells in vitro. On the other hand, expression levels of HA were comparable between B16-F1 and B16-F10 melanoma cells in cryostat sections. These results show that B16-F1 cells express high levels of HA in vitro and in vivo, while B16-F10 cells express high concentrations of HA only in the context of skin tumors. Finally, B16-F10 melanoma cells, but not B16-F1 cells, expressed high concentrations of HA after stimulation with lactate. We propose that components of the tumor microenvironment (e.g., lactate) can induce melanoma cells to express HA and thus acquire an aggressive phenotype.

Animals↗
PubMed2005

Pep-1 as a novel probe for the in situ detection of hyaluronan.

Hyaluronan (HA) is expressed by most tissues, including skin. Localization of HA in the skin is assessed by histology with HA-binding protein (HABP) serving as the probe. Reports have suggested that HA expression in skin is altered in a number of diseases. However, interlaboratory variations in HABP staining profiles, even in normal skin, suggest a need to standardize methods and/or identify new probes. We report the staining patterns of a HA-binding peptide (termed "Pep-1") in human and mouse skin. After acetone fixation, Pep-1 stained HA in the intercellular spaces of the epidermis, whereas staining in the dermis was weak and diffuse in both human and mouse skin. HABP staining of the epidermis and dermis were comparable in human skin but failed to stain the vital epidermis of mouse skin. In human skin, Pep-1 stained the basal, spinous, and granular layers, whereas HABP failed to stain the basal layer. Precipitation of HA in situ resulted in dermal staining but weak staining in the epidermis for HABP and Pep-1. Our results may suggest that Pep-1 is sensitive to HA conformation. Furthermore, Pep-1 may represent a new probe to study HA expression in the skin.

Animals↗
PubMed2005

Immunologic roles of hyaluronan.

Hyaluronan (HA), a large glycosaminoglycan composed of D-N-acetylglucosamine and D-glucuronic acid, is expressed in virtually all tissues and has long been considered to serve as a structural component or filling material in the tissue interstitium (Filler Theory). This idea was revised with the discovery of HA-binding proteins that introduced the concept that HA may also serve as an adhesive substrate for cellular trafficking (Adhesion Theory). Most recently, it has been shown that HA fragments can deliver maturational signals to dendritic cells (DCs) and high molecular weight HA polymers can deliver costimulatory signals to T-cells (Signaling Theory). Thus, HA may represent an important component of the immune system. Recently, we have evaluated the impact of HA on Langerhans cell (LC) maturation and migration using a novel peptide inhibitor of HA function, termed Pep-1 (GAHWQFNALTVR). As skin-specific members of the DC family, LCs are crucial for the initiation of cutaneous immune responses. Local injections of Pep-1 prevented hapten-induced LC migration from the epidermis, providing the first experimental evidence that HA facilitates their emigration. Moreover, Pep-1 also significantly inhibited the hapten-induced maturation of LCs in vivo as assessed by cell morphology, costimulatory molecule expression, and their ability to induce proliferation of allogeneic T-cells. HA therefore has dual functionality to facilitate LC migration and maturation, the two critical events for the initiation of adaptive immune responses. Finally, we have observed that DC-dependent, antigen-specific T-cell proliferation and cytokine secretion is blocked by Pep-1. These results have revealed a previously unrecognized role for HA in antigen presentation. Thus, far from an inert structural biopolymer, HA represents a multifunctional carbohydrate mediator of immune processes.

Animals↗
PubMed2003

Generation and function of reactive oxygen species in dendritic cells during antigen presentation.

Although reactive oxygen species (ROS) have long been considered to play pathogenic roles in various disorders, this classic view is now being challenged by the recent discovery of their physiological roles in cellular signaling. To determine the immunological consequence of pharmacological disruption of endogenous redox regulation, we used a selenium-containing antioxidant compound ebselen known to modulate both thioredoxin and glutaredoxin pathways. Ebselen at 5-20 micro M inhibited Con A-induced proliferation and cytokine production by the HDK-1 T cell line as well as the LPS-triggered cytokine production by XS52 dendritic cell (DC) line. Working with the in vitro-reconstituted Ag presentation system composed of bone marrow-derived DC, CD4(+) T cells purified from DO11.10 TCR-transgenic mice and OVA peptide (serving as Ag), we observed that 1) both T cells and DC elevate intracellular oxidation states upon Ag-specific interaction; 2) ebselen significantly inhibits ROS production in both populations; and 3) ebselen at 5-20 micro M inhibits DC-induced proliferation and cytokine production by T cells as well as T cell-induced cytokine production by DC. Thus, Ag-specific, bidirectional DC-T cell communication can be blocked by interfering with the redox regulation pathways. Allergic contact hypersensitivity responses in BALB/c mice to oxazolone, but not irritant contact hypersensitivity responses to croton oil, were suppressed significantly by postchallenge treatment with oral administrations of ebselen (100 mg/kg per day). These results provide both conceptual and technical frameworks for studying ROS-dependent regulation of DC-T cell communication during Ag presentation and for testing the potential utility of antioxidants for the treatment of immunological disease.

Allergens↗
PubMed2003

Hair follicles serve as local reservoirs of skin mast cell precursors.

Several leukocyte populations normally reside in mouse skin, including Langerhans cells and gammadelta T cells in the epidermis and macrophage and mast cells in the dermis. Interestingly, these skin resident leukocytes are frequently identified within or around hair follicles (HFs), which are known to contain stem cell populations that can generate the epidermal architecture or give rise to the melanocyte lineage. Thus, we reasoned that HFs might serve as a local reservoir of the resident leukocyte populations in the skin. When vibrissal follicles of adult mice were cultured in the presence of stem cell factor (SCF), interleukin 3 (IL-3), IL-7, granulocyte-macrophage colony-stimulating factor, and Flt3 ligand, CD45+/lineage-/c-kit+/FcepsilonRI+ cells became detectable on the outgrowing fibroblasts in 10 days and expanded progressively thereafter. These HF-derived leukocytes showed characteristic features of connective tissue-type mast cells, including proliferative responsiveness to SCF, metachromatic granules, mRNA expression for mast cell proteases-1, -4, -5, and -6, and histamine release on ligation of surface IgE or stimulation with substance P or compound 48/80. These results, together with our findings that HFs contain c-kit+ cells and produce SCF mRNA and protein, suggest that HFs provide a unique microenvironment for local development of mast cells.

Animals↗
PubMed2003

Involvement of hyaluronan in epidermal Langerhans cell maturation and migration in vivo.

BACKGROUND: Langerhans cells (LC) normally reside in the epidermis, where they constantly monitor the integrity of this microenvironment. Skin exposure to reactive haptens or other pro-inflammatory stimuli triggers LC migration to the draining lymph nodes, where they function as potent antigen presenting cells. At the same time, migratory LC exhibit features of mature dendritic cells. OBJECTIVE: The purpose of this study was to determine the potential role(s) of hyaluronan (HA) in the process of LC maturation. METHODS: Using a recently described inhibitor of HA, termed 'Pep-1', we assessed the kinetics of LC migration and maturation in mice. Migration was determined by assessing LC density in epidermal sheets after hapten application. Epidermal LC maturation was evaluated by cell morphology, surface expression of CD86 and allogeneic mixed lymphocyte reactions. RESULTS: Local injections of Pep-1 prevented LC migration from hapten-painted skin almost completely in the first 24 h. On the other hand, LC began to emigrate from the epidermis at 48 h, indicating that Pep-1 simply delayed LC migration by approximately 24 h. Locally injected Pep-1 inhibited hapten-induced LC maturation at 24 h as assessed by morphological changes, CD86 expression levels, and T cell-stimulatory potentials. Strikingly, all maturational changes became detectable at 48 h. CONCLUSION: Pep-1 inhibits LC migration and maturation with the same kinetics, presumably reflecting close association of the two events. Moreover, our results suggest dual functionality of HA to facilitate LC migration and maturation, the two critical events for the initiation of adaptive immune responses in the skin.

Amino Acid Sequence↗
PubMed2003

Keratinocyte ATP release assay for testing skin-irritating potentials of structurally diverse chemicals.

Irritant dermatitis represents innate inflammatory responses to toxic chemicals. We have reported recently that ATP released from chemically injured keratinocytes may serve as a causative mediator for irritant dermatitis. In this study, we examined whether ATP release from keratinocytes would serve as a reliable readout for predicting skin irritating potentials of structurally diverse compounds. A vast majority (19/20) of the tested compounds, i.e., strong and weak irritant chemicals selected from the literature, induced rapid (<10 min) and significant (P<0.05) ATP release from Pam 212 keratinocytes. Two compounds caused no detectable skin inflammation in our standard mouse model, documenting relatively high sensitivity (false negative rate of 0/18) and specificity (false positive rate of 1/20) of our ATP release assay. Selected compounds, primarily those containing phenol residues or hydrophobic hydrocarbon chains, triggered rapid (<10 min) and robust leakage of a fluorescence probe from liposomes, suggesting that lipid bilayers serve as one, but not the only, target moiety on keratinocytes. Not only do our data support the pathogenic role for keratinocyte-derived ATP in irritant dermatitis, they also form the basis for a formal validation study to evaluate the utility of the keratinocyte-based in vitro assay in screening environmental and industrial chemicals.

Adenosine Triphosphatases↗
PubMed2003

Functional roles of hyaluronan in B16-F10 melanoma growth and experimental metastasis in mice.

Although hyaluronan (HA), a high molecular weight glycosaminoglycan, is generally believed to regulate tumor growth, invasion, and metastasis, functional roles of HA have only been speculated indirectly from the outcome of blocking HA receptors (e.g., CD44). Using a phage display technique, we recently developed a synthetic peptide (GAHWQFNALTVR; Pep-1) that binds to and inhibits the function of HA. In this study, we have used Pep-1 to determine whether HA directly regulates the behavior of tumor cells. B16-F10 melanoma cells, which constitutively expressed CD44, showed significant adhesion to HA-coated plates, and this adhesion was blocked almost completely either by neutralizing antibodies against CD44 or by Pep-1. These results imply that CD44 is the primary HA receptor mediating the adhesive interaction of the melanoma cells with HA substrates. In contrast, Pep-1 failed to inhibit in vitro proliferation of B16-F10 melanoma cells or the in vitro growth of the cells after s.c. inoculation in mice. Importantly, single injection of Pep-1 significantly reduced the incidence of lung metastasis of i.v. inoculated melanoma cells and prolonged the survival of the tumor-bearing animals. These results suggest a direct contribution of HA to one or more steps in the initial seeding of melanoma cells in the lung microenvironment. Our observations also introduce a new concept that synthetic inhibitors of HA may represent unique tools for studying the roles of HA in tumor biology and perhaps a new class of therapeutic reagents.

Animals↗
PubMed2002

Synthesis and surface expression of hyaluronan by dendritic cells and its potential role in antigen presentation.

Hyaluronan (HA) is a large glycosaminoglycan consisting of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. HA is known to act as a filling material of extracellular matrices and as an adhesive substrate for cellular migration. Here we report that dendritic cells (DC) express mRNAs for HA synthases and hyaluronidases, actively synthesize HA, and display HA on their surfaces. Interestingly, HA expression levels on DC were not significantly altered by their maturation states. With respect to physiological function, three specific HA inhibitors, i.e., bovine proteoglycan, a 12-mer HA-binding peptide (GAHWQFNALTVR) termed Pep-1, and an oligomeric Pep-1 formulation, all interfered with DC-induced activation of CD4(+) T cells isolated from DO11.10 TCR transgenic mice. For example, Pep-1 oligomer efficiently inhibited DC-dependent cluster formation, IL-2 and IFN-gamma production, and proliferation by DO11.10 T cells in vitro without affecting the viabilities of DC or T cells, DC function to uptake exogenous proteins, or DC-T cell conjugate formation at earlier time points. These observations suggest a paracrine mechanism by which DC-associated HA facilitates some of the late changes in T cell activation. Although T cells constitutively expressed mRNAs for HA synthases and hyaluronidases, their surface HA expression became detectable only after activation. Oligomeric Pep-1 and bovine proteoglycan both inhibited mitogen-triggered T cell activation in the absence of DC, suggesting an autocrine mechanism by which HA expressed by T cells assists their own activation processes. Finally, adoptively transferred DO11.10 T cells showed progressive mitosis when stimulated with Ag-pulsed DC in living animals, and this clonal expansion was inhibited significantly by administration of Pep-1 oligomer. Our findings may introduce a new concept that relatively simple carbohydrate moieties expressed on DC and perhaps T cells play an important immunomodulatory role during Ag presentation.

Adjuvants, Immunologic↗