Liesbet Paemen

Resistance of young gelatinase B-deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions.

Regulated expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) plays a role in various physiological processes. To determine in vivo how unbalanced expression of these factors can promote or affect the course of pathologies, we knocked out the mouse gelatinase B gene by replacing the catalytic and zinc-binding domains with an antisense-oriented neomycin resistance gene. Adult gelatinase B-deficient mice and wild-type controls could be induced to develop experimental autoimmune encephalomyelitis (EAE) with similar scores for neurologic disease, blood-brain barrier permeability, and central nervous system histopathology. However, whereas diseased control animals showed necrotizing tail lesions with hyperplasia of osteocartilaginous tissue, adult gelatinase B-deficient mice were resistant to this tail pathology. Gelatinase B-deficient mice younger than 4 weeks of age were significantly less susceptible to the development of EAE than were age matched controls and, even as they aged, they remained resistant to tail lesions. These data illustrate that gelatinase B expression plays a role in the development of the immune system and that, in ontogenesis, the propensity to develop autoimmunity is altered by the absence of this MMP.

The expression of tissue-type plasminogen activator, matrix metalloproteases and endogenous inhibitors in the central nervous system in multiple sclerosis : Comparison of stages in lesion evolution

The expression of tissue-type plasminogen activator (t-PA) and a number of metalloproteases as well as plasminogen activator inhibitor-1 (PAI-I) and tissue inhibitor of metalloproteases-1 (TIMP-1) was analyzed in the central nervous system (CNS) of normal control and multiple sclerosis (MS) cases by immunohistopathology. The expression of t-PA was detectable only in the blood vessel matrix in control white matter, but positive infiltrating mononuclear cells were also observed in MS white matter and primary lesions. In active plaques this pattern converted to strong positivity of foamy macrophages in areas of demyelination, declining in chronic lesions. In general PAI-1 expression paralleled that of t-PA. Gelatinase A and B were detected predominantly in astrocytes and microglia throughout normal control white matter, with additional positive mononuclear cells in perivascular cuffs in MS white matter. In the demyelinating lesion there is widespread prominent expression of gelatinase B in reactive astrocytes and macrophages, which persists in astrocytes in the chronic lesion. TIMP-1 was also present in the vessel matrix and in lesional macrophages. These observations on the coexpression of enzymes and inhibitors of the matrix degrading cascade in CNS tissue pinpoint t-PA, a -limiting enzyme, and gelatinase B as therapeutic targets in MS.

The gelatinase inhibitory activity of tetracyclines and chemically modified tetracycline analogues as measured by a novel microtiter assay for inhibitors

A quantitative nonisotopic solution assay for gelatinases and inhibitors was developed using biotinylated gelatin as enzyme substrate. In this assay, residual biotinylated substrate is sandwiched between avidin-coated plates and streptavidin-peroxidase and is quantified by the peroxidase reaction. This assay was useful for measuring gelatinase activities and defining the activities of gelatinase inhibitors. When 23 tetracycline analogues were compared, significant differences in gelatinase B inhibition were found between various compounds. 4-epioxytetracycline base, 4-epichlortetracycline, meclocyclinesulfosalicylate, and unmodified metacycline and minocycline proved to be the most potent gelatinase B (EC 3.4.24.35) inhibitors. The gelatinase B inhibitory activity of tetracyclines was clearly dissociated from their antimicrobial activity. The effect of high-molecular-weight inhibitors, such as monoclonal antibodies, was also demonstrable in the microtiter plate assay. In view of the pathophysiological function of gelatinases, the definition of gelatinase inhibitors with known efficacy, safety, and side effects is crucial for the treatment of diseases such as rheumatoid arthritis and multiple sclerosis. Particular tetracyclines fulfil these criteria and the described assay is useful for defining other gelatinase-inhibiting lead compounds.

Lom-AG-myotropin: A novel myotropic peptide from the male accessory glands of Locusta migratoria

A myotropic peptide, termed Lom-AG-myotropin, was isolated from extracts of 4400 accessory gland complexes of males of the locust, Locusta migratoria; the following sequence was derived: Gly-Phe-Lys-Asn-Val-Ala-Leu-Ser-Thr-Ala-Arg-Gly-Phe-NH2. This sequence is completely different from all presently known myotropic peptides from Locusta or other insects. The Lom-AG-myotropin is active on the oviduct and hindgut of Locusta migratoria and Leucophaea maderae. The stimulatory activity is, in both insects, 1000 times greater on the oviduct than on the hindgut, suggesting a specificity for the oviduct.

Synergistic induction of MCP-1 and -2 by IL-1beta and interferons in fibroblasts and epithelial cells.

Monocyte chemotactic protein (MCP)‐1 and MCP‐2, two closely related CC chemokines, are important mediators of monocyte and lymphocyte migration. These chemokines are secreted by various normal cell types, including fibroblasts, epithelial cells, and leukocytes, as well as by tumor cells. After stimulation with different cytokines and cytokine inducers the MCP‐2 production levels are always lower than those of MCP‐1. In human diploid fibroblasts cytokines differentially regulate chemokine induction, interleukin (IL)‐1β and interferon (IFN)‐γ being potent stimuli of MCP‐1 and MCP‐2, respectively. Co‐stimulation of fibroblasts by 10 U/mL IL‐1β and 20 ng/mL IFN‐γ resulted in a synergistic induction of MCP‐2, whereas the combined effect on MCP‐1 and IL‐6 production was rather additive. These findings were confirmed at the mRNA level by Northern blot analysis. In contrast, in human MG‐63 fibroblastoid cells and HEp‐2 epithelial cells, selected for their poor responsiveness to IL‐1β and IFN‐γ, MCP‐2 as well as MCP‐1 and IL‐6 were synergistically induced, yielding protein levels that were increased 3‐ to 30‐fold above the additive levels. When IFN‐β was used as a co‐stimulant of IL‐1β, a similar synergistic induction of MCP‐1 and MCP‐2 was measured both at the protein and the mRNA level. It can be concluded that, when synergy occurred, the MCP‐1 and MCP‐2 expression levels reached a comparable maximum, indicative for an equal contribution of these chemokines in normal and pathological conditions. J. Leukoc. Biol. 63: 364–372; 1998.

Prevention of acute autoimmune encephalomyelitis and abrogation of relapses in murine models of multiple sclerosis by the protease inhibitor D-penicillamine

Thein vitro activity of gelatinase B, an enzyme whose appearance in the cerebrospinal fluid is associated with inflammatory diseases of the central nervous system, was dose-dependently inhibited by the antirheumatic D-penicillamine. Inhibition of gelatinase B in electrophoretically pure preparations and in cell culture supernatants and human body fluids was obtained at dosages reached in the circulation of patients treated with a peroral dosis of 750mg D-penicillamine per day. In mice, developing acute demyelination, D-penicillamine significantly reduced the mortality and morbidity rates of experimental allergic encephalomyelitis (EAE). In chronic relapsing EAE in Biozzi AB/H mice, an animal model for relapses in multiple sclerosis (MS), it attenuated the exacerbations, even when the treatment was started after the primary full-blown disease had developed. We infer protease inhibition as the mechanism of action of D-penicillamine and suggest that its use may be effective as peroral treatment for MS.

Co-localization of locustamyotropin- and pheromone biosynthesis activating neuropeptide-like immunoreactivity in the central nervous system of five insect species

Abstract 1. 1. Myotropin I of Locusta and Pheromone Biosynthesis Activating Neuropeptide (PBAN) of Heliothis share the same carboxyterminal pentapeptide FSPRL-amide. 2. 2. Immunostaining revealed colocalization in cells and axons in the central nervous system, especially in the suboesophageal ganglion, of Locusta, Periplaneta, Leucophaea, Neobellieria (Sarcophaga) and Mamestra. 3. 3. Following preabsorption with synthetic FSPRL-amide, the PBAN antiserum continued to immunostain cells in Mamestra and Neobellieria only. The preabsorbed Lom-MT I antibody did not yield any positive reaction. 4. 4. Our results indicate that the functional epitope FXPRL-amide is widespread among insect orders. Its distribution in the nervous system seems to be rather similar in all investigated species.

Localization of Lom-AG-myotropin I-like substances in the male reproductive and nervous tissue of the locust, Locusta migratoria

SummaryLom-AG myotropin I (Lom-AG-MTI) was the first peptide to be isolated from the male accessory reproductive glands of the locust, Locust migratoria. It shows no sequence similarity to any of the peptides identified from vertebrate or invertebrate tissues. A polyclonal antiserum was used to localize Lom-AG-MTI-like material in the male reproductive system and nervous system of the locust. Immunoreactivity was found in two of the hyaline gland tubules. In the brain, cell bodies were detected in the proto- and deuterocerebrum as well as the frontal ganglion. Nerve fibers were stained in the neuropils of the brain and throughout the labial nerves into the recurrent nerve. Thoracic and last abdominal ganglia contained neurons which could be stained with Lom-AG-MTI antiserum. The pronounced reactivity in the central nervous system suggests a possible neuroregulatory function of the peptide.

Leukocyte recruitment by monocyte chemotactic proteins (MCPs) secreted by human phagocytes

Phagocyte recruitment is an important immunological phenomenon in inflammation and cancer. A large family of selective chemotactic cytokines, designated chemokines, has recently emerged. Interleukin-8 (IL-8) is the prototype of such neutrophil activating factors, whereas MCP-1 is a well studied monocyte chemotactic protein. In vitro chemotactic assays were used to isolate and identify natural chemokines from mononuclear phagocytes and tumor cells. Additional new chemotactic proteins (MCP-2, MCP-3) attracting monocytes were also discovered by these methods. All chemokines are structurally related and show affinity for heparin. MCP-1, -2 and -3 have a comparable specific activity in monocyte chemotaxis assays. Specific and sensitive radioimmunoassays for MCP-1 and IL-8 were developed to study the regulation of their secretion by leukocytes. Monocytes or monocyte tumor cells produce MCP-1 and/or IL-8 in response to cytokines, virus, double stranded RNA, bacterial endotoxin, mitogen or phorbol ester. Granulocytes were found to secrete only minor amounts of MCP-1 and IL-8.

Expression of gelatinase B in trachomatous conjunctivitis

BACKGROUND/AIMS Gelatinase B is a matrix metalloproteinase involved in extracellular matrix (ECM) breakdown often associated with scarring and other pathological disorders. It was investigated whether gelatinase B is involved in the pathogenesis of ECM degradation associated with trachomatous conjunctivitis. METHODS Conjunctival biopsy specimens obtained from six patients with active trachoma, six patients with active vernal keratoconjunctivitis (VKC), and seven control subjects were studied. Immunohistochemical techniques and a specific monoclonal antibody against human gelatinase B were used, and a monoclonal antibody against macrophage CD68 to identify mononuclear cells with gelatinase B immunoreactivity. In addition, quantitative zymography was used to compare the activity of gelatinase B in conjunctival biopsy specimens from seven patients with active trachoma and seven control subjects. RESULTS Gelatinase B was detected by immunohistochemistry only in polymorphonuclear cells located in the vascular lumens in three normal conjunctival biopsy specimens. In all trachoma specimens and in five VKC specimens, gelatinase B was localised in monocyte/macrophage cells, positive for the CD68 marker, and in polymorphonuclear cells. The majority of the latter cell type was located in intravascular spaces. Compared with VKC specimens, trachoma specimens showed significantly more immunoreactive gelatinase B monocyte/macrophage cells (52.3 (21.9)v 8.2 (6.4); p <0.001) and polymorphonuclear cells (23.2 (14.2) v 6.3 (5.4); p = 0.013). Activated macrophages with giant cell morphology clearly stained with the gelatinase B specific monoclonal antibody were observed in trachoma specimens. Zymography revealed that gelatinase B levels in trachoma specimens were significantly higher than the levels found in normal conjunctiva (1739.6 (1078.3)v 609.3 (395.9) scanning units; p = 0.0127). CONCLUSIONS The increased activity of gelatinase B and numbers of inflammatory cells containing gelatinase B in trachoma specimens suggest that this enzyme plays a part in the pathogenesis of conjunctival scarring in trachoma.