Andrew J.H. Gearing

Gelatinase B and TIMP-1 are regulated in a cell- and time-dependent manner in association with neuronal death and glial reactivity after global forebrain ischemia

Matrix metalloproteinases (MMPs) belong to a large family of endopeptidases that regulate the pericellular environment through the cleavage of protein components of the extracellular matrix, membrane receptors and cytokines. MMP activity is controlled by the multifunctional tissue inhibitors of metalloproteinases (TIMPs). Proteases and their inhibitors are critically involved in developmental and pathological processes in numerous organs, including the brain. Global transient cerebral ischemia induces selective delayed neuronal death and neuroinflammation. We compared, in discrete vulnerable and resistant areas of the ischemic rat hippocampus, the kinetics and cellular distribution of gelatinase B and its principal inhibitor TIMP‐1 and we assessed by in situ zymography, the net gelatinolytic activity at the cellular level. We show that gelatinases are expressed and active in neurons, suggesting that MMPs play a role in maintaining neural homeostasis. In the ischemic rat brain, expression and activity of gelatinase B, and expression of TIMP‐1 are altered in a time‐, region‐ and cell‐dependent manner. Gelatinase B is induced first in reactive microglia and subsequently in reactive astrocytes. In situ, increases in gelatinase activity accompanied the progression of neuronal death and glial reactivity. Our results suggest that MMPs and TIMPs are involved in cell viability and tissue remodelling in the ischemic brain, and reinforces the idea that the MMP/TIMP system contributes both to neuronal demise and tissue repair in the context of glial reactivity.

Cytokines in skin lesions of psoriasis

Cytokine levels were compared in aqueous extracts of stratum corneum from psoriatic lesions and normal heel. Samples from heel contained high levels of interleukin-1 alpha (IL-1 alpha) and beta measured in immunoassays, although only the IL-1 alpha was biologically active. No other cytokines could be detected in heel samples. Interleukin-1 (IL-1) levels were dramatically reduced in lesional samples. A neutrophil chemoattractant was found in all lesional extracts, and was demonstrated to be mainly interleukin-8 (IL-8) using a specific neutralizing antiserum. Tumor necrosis factor alpha (TNF-alpha) and beta (TNF-beta), and interferon alpha (IFN-alpha) and gamma (IFN-gamma) were detected in lesional extracts using immunoassays, however, no equivalent biological activities could be detected. Interleukins 2 (IL-2), 4 (IL-4), and 6 (IL-6), granulocyte and granulocyte/macrophage colony stimulating factor (GM-CSF), could not be detected in any samples. IL-8 is therefore the only biologically active cytokine shown in this study to be elevated in psoriatic lesional extracts, and may therefore play a role in the pathogenesis of the disease.

Induction of in vitro human lymphocyte migration by interleukin 3, interleukin 4, and interleukin 6.

The effects of interleukin 3 (IL 3), IL 4, IL 6, and interferon-gamma (IFN-gamma) on lymphocyte migration have been investigated and compared with those of transforming growth factor-beta 1 (TGF-beta 1), granulocyte colony stimulating factor (GCSF), and macrophage colony stimulating factor (MCSF). Potent, temperature-dependent stimulation of lymphocyte migration was obtained in response to IL 3 and IL 4 (ED50 less than 10(-11) M and less than 10(-13) M, respectively) and this migration was abolished in the presence of 3 micrograms ml-1 cytochalasin B. IL 6 and IFN-gamma were less active (ED50 greater than or equal to 10(-9) M and greater than or equal to 10(-8) M, respectively), maximal migration in response to IFN-gamma being only 30% above background as compared with approximately 250% for IL 3 and IL 4. TGF-beta 1, GCSF, and MCSF failed to stimulate lymphocyte migration in doses similar to those used for IL 3, IL 4, and IL 6. The presence of antisera to IL 3, IL 4, and IL 6 specifically inhibited lymphocyte migration induced by the corresponding cytokines (IC50 values being 1/10,000, greater than 1/30,000, and greater than 1/30,000 dilution of antibody, respectively). Cross-desensitization experiments using IL 3 and IL 4 demonstrated that neither IL 3 nor IL 4 were able to stimulate dose-related lymphocyte migration in cells preincubated with IL 3. Cells preincubated with IL 4 were only stimulated by a supraoptimal concentration of IL 4 (10(-11) M). The induction of lymphocyte migration by IL 3, IL 4, and IL 6 therefore appears to be a specific and potentially important effect of these cytokines. Cross-desensitization of lymphocytes by IL 3 and IL 4 raises the possibility that the induction of lymphocyte migration by these cytokines may occur through a common postreceptor signal transduction mechanism.

Extracellular matrix-mediated chemotaxis can impede cell migration

Cells use a combination of changes in adhesion, proteolysis and motility (directed and random) during the process of migration. Proteolysis of the extracellular matrix (ECM) results in thecreation of haptotactic gradients which cells use to move in a directed fashion. The proteolytic creation of these gradients also results in the production of digested fragments of ECM. In this study we show that in the human fibrosarcoma cell line HT1080, matrix metalloproteinase–2(MMP–2)–digested fragments of fibronectin exert a chemotactic pull stronger than that of undigested fibronectin. During invasion, this gradient of ECM fragments is established in the wake of an invading cell, running counter to the direction of invasion. The resultant chemotactic pull is anti–invasive, contrary to the traditional view of the role of chemotaxis in invasion. Uncontrolled ECM degradation by high concentrations of MMP can thus result in steep gradients of ECM fragments, which run against the direction of invasion. Consequently, the invasive potential of a cell depends on MMP production in a biphasic mannerimplying that MMP inhibitors will upregulate invasion in high–MMPexpressing cells. Hence the therapeutic use of protease inhibitors against tumours expressing high levels of MMP could produce an augmentation of invasion.

The use of Tween 20 alone as a blocking agent for immunoblotting can cause artefactual results

The use of Tween 20 as a suitable blocking agent in immunoblotting studies was evaluated by screening a panel of monoclonal antibodies (MoAbs) against a selection of blotted proteins which were unrelated to the antigens used to raise the MoAbs. Using Tween 20 alone to block the nitrocellulose membranes clear reactions were observed between the panel of MoAbs and several components of the blotted protein mixture. In contrast, when haemoglobin was used to block the membranes such reactions were not observed. In the absence of added protein the use of Tween 20 alone as a blocking agent for immunoblotting appears to lead to false positive reactions by non-specific antigen-antibody complexes.

Skin exudate levels of interleukin 6, interleukin I and other cytokines in mycosis fungoides

The role of locally released cytokines in inducing lymphocyte activation and infiltration in the skin lesions of mycosis fungoides has been investigated. The levels of selected cytokines were measured in chamber fluid samples from lesional and control skin. Biologically active interleukin 6 was significantly elevated in lesional samples and a recombinant form of this cytokine was shown to induce lymphocyte migration in an in vitro assay. Biologically active interleukin 1 was detected in all control chamber fluid samples. Significantly reduced levels of this cytokine were present in lesional samples, which may be the result of the release of preformed material. Interleukin 2 and tumour necrosis factor activity, and γ interferon and granulocyte macrophage colony‐stimulating factor immunoreactivity, were not detectable in any of the samples. Interleukins 1 and 6 may play a role in the pathogenesis of the lesional lymphocyte infiltrates in mycosis fungoides.

Interleukin 2 stimulates T cell proliferation using a calcium flux

A preparation enriched in rat interleukin 2 caused enhanced DNA synthesis in an interleukin 2-dependent mouse cytotoxic T cell line, in lectin transformed mouse splenocytes and in rat thymocytes. The enhanced proliferation due to interleukin 2 could be abrogated by chelating calcium from the culture medium or blocking calcium entry into the cells. Compounds which interfere with the function of calmodulin also inhibited proliferation. The addition of interleukin 2 to IL-2 dependent cells caused an increase in the intracellular concentration of calcium ions, as measured using Quin 2. The requirement for IL-2 by blasts and thymocytes could be replaced by calcium ionophore. The results implicate a calcium flux as an essential component of the action of interleukin 2 on its target cells.

Human B cell proliferation is stimulated by interleukin 2

The proliferation of human B cells was studied for response to interleukin 2 (IL-2) produced in Escherichia coli using recombinant DNA technology. The IL-2 was found to be an homogenous preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the anti-IL-2 monoclonal antibody DMS-1. IL-2 was found to stimulate B cell proliferation. Activation of the B cells using anti-IgM antibodies increased this response. Resting T cells from the same donors were found to be less reactive to IL-2. The results suggest that human B cell proliferation can be stimulated by IL-2 alone.

Production of polyclonal and monoclonal antibodies to human granulocyte colony-stimulating factor (GCSF) and development of immunoassays

Murine monoclonal antibodies and a sheep polyclonal antiserum against recombinant human granulocyte colony-stimulating factor (GCSF) have been produced. These have been used to develop an immunoassay which can detect 250 pg/ml (25 U) of both natural and recombinant human GCSF. The assay involves forming a complex between GCSF and a monoclonal anti-GCSF, binding of the complex to microtitre wells coated with sheep anti-GCSF and detection of the bound complex with 125I-labelled sheep anti-mouse IgG. Unlike the classical bone marrow assay and other cell line based bioassays for GCSF, the immunoassay was specific for the cytokine, showing no cross-reactivity with GM-CSF, IL-6, IL-3 or IL-1 alpha and -beta. The assay does not exhibit interfering matrix effects when used for the estimation of human GCSF in serum.

CHEMOTACTIC CYTOKINES IN INFLAMMATORY SKIN DISEASE

The production of cytokines in human skin in vivo has been investigated and found to be more selective than studies in vitro have suggested. Thus, stratum corneum samples from the skin lesions of psoriasis contain interleukin 8 (IL-8)-like material, but assay for a range of other compounds suggested that no other defined, biologically active cytokine was present in increased levels when compared with those in control heel stratum corneum samples. Selective cytokine release was also found on analysis of chamber fluid samples from the skin lesions of the cutaneous T-cell lymphoma, mycosis fungoides. Assay of normal epidermal samples has shown the presence of biologically active amounts of IL-1 like material, chromatographic purification and the use of neutralizing antibodies indicating the presence of IL-1α but negligible IL-1β activity in normal heel stratum corneum extracts. No other biologically active cytokine has been detected in normal skin. The IL-1α-like material recoverable from normal human epidermis possesses potent inflammatory properties when injected intradermally, but appears not to be biologically available under normal in vivo conditions, possibly through intracellular retention in keratinocytes, membrane association or control by an inhibitor. The release of preformed IL-1 following membrane perturbation or other events may constitute a primary mechanism for the induction of inflammation in human skin. For reasons to be outlined below, IL-1 may be less important in the maintenance of chronic inflammatory changes, at least in psoriasis, IL-8 possibly playing a more significant role.