Detection of macrophage migration inhibitory factor by monoclonal antibody in Sézary syndrome.

We have reported previously on the generation of a monoclonal antibody against human macrophage migration inhibitory factor (MIF), which is a mediator of cellular immunity. Macrophage migration inhibitory factor activity in the migration assay was closely correlated with antibody reactivity. Using this antibody called 1C5/B, we are now able to study the expression of MIF in situ. Here, we report on the detection of MIF in blood lymphocytes and skin of a patient with a leukemic cutaneous T-cell lymphoma with the characteristics of Sézary syndrome. Ninety percent of the patient's Ficoll Hypaque-isolated peripheral white blood cells were of the helper phenotype. By conventional immunoperoxidase method, 94% reacted strongly positive with the antibody 1C5/B. In contrast, using the immunofluorescence method only 25% reacted positive. This indicates that the majority of the tumor cells did not express the molecule on their membrane but only in the cytoplasma. No other marker, such as interleukin 2 receptor, HLA-DR antigen, or interferon-gamma could be related to the expression of MIF. Also the cellular infiltrate in the skin was composed mainly of T helper cells and reacted positive with 1C5/B. As less than 3% of normal blood lymphocytes reacted with 1C5/B we suggest that the conversion to positivity may be a characteristic feature of the leukemic T-cell phenotype in Sézary syndrome.

Biosynthesis of acid alpha-glucosidase in late-onset forms of glycogenosis type II (Pompe's disease).

Cultured human skin fibroblasts from control persons and from patients with the generalized and late-onset forms of Pompe's disease were labelled with radioactive leucine and the incorporation of radioactivity into acid alpha-glucosidase and cathepsin D was analysed by immunoprecipitation, gel electrophoresis and fluorography. When the labelling was carried out for 6-12 h in the presence of NH4Cl, the labelling of secreted alpha-glucosidase relative to that of secreted cathepsin D in fibroblasts from patients with the late-onset form of Pompe's disease was less than 15% of that in fibroblasts from control persons. However, when the fibroblasts were labelled for less than 1 h, the relative rate of incorporation of radioactivity into acid alpha-glucosidase was rather similar in the two types of fibroblasts. In fibroblasts from patients with the generalized form of Pompe's disease no incorporation of radioactivity into acid alpha-glucosidase could be detected.

Differentiation of monocytes in vitro: Influence of different types of substances.

The monocyte is one of the cell types activated in inflammatory processes to leave the blood and to enter the site of tissue inflammation through the endothelium as part of the cellular infiltrate. In tissue, monocytes differentiate to macrophages. This process is characterized by the expression of differentiation antigens, which can be monitored by monoclonal antibodies. A set of monoclonal antibodies has been used to design a test to analyse the ability of substances to influence the pattern of antigen expression during macrophage differentiation. Irritant substances stimulate the enhanced expression of an 'acute marker', which indicates the infiltration of macrophages into acutely inflamed tissues. Anti-inflammatory substances stimulate the enhanced expression of a 'middle marker' associated with the down-regulatory phase of macrophages. The stimulation of the 'acute marker' is positively correlated with the potential of substances and products to induce or elicit adverse skin reactions.

Immunocompetent cells for in vitro screening of skin irritation.

The present studies were aimed at evaluating procedures for assessing the immunmodulatory effects of chemicals and preparations on macrophage differentiation and lymphocyte proliferation in cell cultures. The effects of 10 drugs and anti-inflammatory agents were monitored by determining thymidine incorporation into phytohaemagglutinin (PHA)-stimulated T cells in the lymphocyte transformation test (LTT) and the expression of two surface antigens on macrophages in the macrophage differentiation assay (MDA). One antigen was found on macrophages in acute inflamed tissue. The other was detected on those found in recovering tissue. These parameters were compared with mean skin irritation scores for 12 known cosmetic products from epicutaneous patch testing. Finally, these parameters were also used to study six cosmetic test formulae with unknown irritation potentials subjected to blind testing during phase 2 of the "CTFA Evaluation of Alternatives Program". Immunosuppressive agents were detected in both systems. Agents, thought to be pro-inflammatory, were monitored in the MDA by the acute inflammation marker. Skin irritation scores of known preparations correlated well with those of expressed acute inflammation markers in the MDA (r(s) = 0.714), but no clear relationship was detectable in the LTT. In contrast one of the CTFA samples tested blind revealed a strong response in both tests. The roll-on antiperspirant stimulated T-cell proliferation and induce a strong expression of the acute inflammation marker on macrophages. Based on these findings further studies are in progress to evaluate the usefulness of these in vitro tests for predicting dermal irritation.

In vitro model for contact sensitization: I. Stimulatory capacities of human blood-derived dendritic cells and their phenotypical alterations in the presence of contact sensitizers.

Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) located in many non-lymphoid tissues and a specialized form of DC-the Langerhans cell (LC)-is found in the skin. The functionality of LC as APC is crucial for the induction of an allergic contact dermatitis. For a long time LC research has been hampered by the limiting numbers of functionally active LC that could be isolated from human skin. The addition of GM-CSF and IL-4 to the non-adherent fraction of mononuclear cells from peripheral blood generated a large amount of CD1a(+) HLA-DR(+) DC. These in vitro-generated DC exhibited the morphology, phenotype and autologous T-lymphocyte stimulating capacity of the human DC/LC system. We had tested phenotypical alterations of in vitro-generated DC under the influence of subtoxic concentrations of different chemicals and contact sensitizers. In vitro stimulation with the contact sensitizers urushiol, primin, C10-and C11-primin analogues, alantolactone, isoalantolactone and NiSO(4) resulted in a decrease of HLA-DR expression on the surface of these cells if the incubation period did not exceed 3 hr. Incubation with irritants like sodium lauryl sulfate (SLS) and benzalkonium chloride did not change or increase the HLA-DR surface expression under these conditions. With regard to the adhesion molecule ICAM-1, there was no clear difference between irritants and contact sensitizers. But based on the alteration of HLA-DR expression of dendritic cells under short-term exposure conditions, there was a clear-cut difference between irritants and contact sensitizers. In summary, this system can be used to discriminate between contact sensitizers and irritants.

Coenzyme Q10, a cutaneous antioxidant and energizer.

The processes of aging and photoaging are associated with an increase in cellular oxidation. This may be in part due to a decline in the levels of the endogenous cellular antioxidant coenzyme Q10 (ubiquinone, CoQ10). Therefore, we have investigated whether topical application of CoQ10 has the beneficial effect of preventing photoaging. We were able to demonstrate that CoQ10 penetrated into the viable layers of the epidermis and reduce the level of oxidation measured by weak photon emission. Furthermore, a reduction in wrinkle depth following CoQ10 application was also shown. CoQ10 was determined to be effective against UVA mediated oxidative stress in human keratinocytes in terms of thiol depletion, activation of specific phosphotyrosine kinases and prevention of oxidative DNA damage. CoQ10 was also able to significantly suppress the expression of collagenase in human dermal fibroblasts following UVA irradiation. These results indicate that CoQ10 has the efficacy to prevent many of the detrimental effects of photoaging.

Biosynthesis and maturation of arylsulfatase B in normal and mutant cultured human fibroblasts.

The biosynthesis of arylsulfatase B in normal and mutant human skin fibroblasts was studied by metabolic labeling with radioactive amino acids, monosaccharides, or 32Pi and by specific immunoprecipitation followed by polyacrylamide gel electrophoresis and fluorography. Three major polypeptides with apparent molecular weights of 47,000, 40,000, and 31,000 were found intracellularly and one of 64,000 in the medium. Pulse-chase labeling and uptake experiments showed that arylsulfatase B synthesized and secreted as a 64,000 precursor was intracellularly processed within less than 24 h via short lived intermediates to two different forms. Form I (chains of 47,000 and 11,500) was labeled earlier and was about twice as stable as form II (chains of 40,000 and 31,000). The secreted 64,000 precursor and the 40,000 chain of form II contained oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H. In the other chains mainly cleavable and phosphorylated oligosaccharides were found. Arylsulfatase B activity was associated with the 64,000 precursor and with form I, but not with form II. Fibroblasts of four patients with the severe form of mucopolysaccharidosis type VI, which were deficient in arylsulfatase B activity, synthesized and secreted the 64,000 precursor at a normal rate. This precursor, however, had little if any catalytic activity and one of its mature forms (I) was rapidly degraded.

Synthesis and stability of arylsulfatase A and B in fibroblasts from multiple sulfatase deficiency.

Fibroblasts from patients with multiple sulfatase deficiency were analyzed for activities of arylsulfatase A and B, iduronate 2-sulfatase and sulfamatase. A group of patients (group I) severely deficient in all sulfatases (residual activities less than or equal to 10% of control) were differentiated from patients (group II) with residual sulfatase activities of up to 90% of control. The synthesis and stability of arylsulfatase A and B were determined in pulse-chase labelling experiments. The apparent rate of synthesis of arylsulfatase A and B varied from 30% to normal in both fibroblasts from group I and II multiple sulfatase deficiency. In group I the molecular activity of the arylsulfatase A and B was more than 10-fold lower than in control fibroblasts. In group II the molecular activity of the arylsulfatase A was twofold to threefold lower and that of arylsulfatase B half of normal. In fibroblasts of both groups the stability of arylsulfatase A polypeptides was significantly diminished. For arylsulfatase B the instability was restricted to the mature 47000-Mr polypeptide and was variable within both groups. These results demonstrate that multiple sulfatase deficiency is a heterogeneous disorder, in which the primary defects can impair both the catalytic properties and the stability of sulfatases.

Multiple sulfatase deficiency: degradation of arylsulfatase A and B after endocytosis in fibroblasts.

Multiple sulfatase deficiency can be classified into group I with severe and group II with moderate deficiencies in sulfatases. In fibroblasts in both groups the stability of arylsulfatase A and of the 47000-Mr form of arylsulfatase B is decreased [F. Steckel, A. Hasilik & K. von Figura (1985) Eur. J. Biochem. 151, 141-145]. After endocytosis in control fibroblasts or those from multiple sulfatase deficiency, arylsulfatase A and B derived from the latter were subjected to enhanced degradation in both types of recipient cells. The degradation was closely linked in time to endocytosis. Whereas instability of arylsulfatase A derived from different cell lines from multiple sulfatase deficiency was comparable, a marked heterogeneity was observed for the instability of the 47000-Mr polypeptide of arylsulfatase B. Each of the cell lines from multiple sulfatase deficiency synthesized arylsulfatase A and B polypeptides with normal and with decreased stability. Treatment with benzyloxycarbonyl-Phe-Ala-CHN2, an inhibitor of cysteine proteinases, stabilized arylsulfatase A polypeptides and partially restored arylsulfatase A activity in group II fibroblasts. The inhibitor had no protective effect on the 47000-Mr polypeptide or the activity of arylsulfatase B. The bearing of these findings on the yet unknown primary defect in multiple sulfatase deficiency is discussed.

Juvenile and adult metachromatic leukodystrophy: partial restoration of arylsulfatase A (cerebroside sulfatase) activity by inhibitors of thiol proteinases.

Arylsulfatase A polypeptides were examined in cultured fibroblasts from a patient with juvenile metachromatic leukodystrophy and three patients with the adult form of the disease, with the aid of metabolic labeling and immunoprecipitation. The mutant cells were severely deficient in the arylsulfatase polypeptides. The apparent rate of synthesis, however, as estimated from the secretion of polypeptides or activity by cells incubated in the presence of 10 mM NH4Cl was 20-50% of control. In the absence of NH4Cl, the mutant enzyme was rapidly degraded upon transport into lysosomes. In the presence of inhibitors of thiol proteinases arylsulfatase A polypeptides were partially protected from degradation, and the catalytic activity of arylsulfatase A was increased. In addition, the treatment partially corrected the capacity of the cells to degrade cerebroside sulfates. Inhibitors of thiol proteinases may be of therapeutic value in variants of metachromatic leukodystrophy, in which an unstable arylsulfatase A is synthesized.

Two allelic forms of human arylsulfatase A with different numbers of asparagine-linked oligosaccharides.

The biosynthesis of arylsulfatase A in human skin fibroblasts was studied by labeling cells and isolating arylsulfatase A using immune precipitation and polyacrylamide gel electrophoresis under denaturing and reducing conditions. Arylsulfatase A was synthesized as precursor polypeptides of 62 kDa or 59.5 kDa. Cell lines synthesizing either or both polypeptides were found. The results of a family study were consistent with the assumption that the two arylsulfatase A polypeptides are of allelic nature. In various heterozygous cell lines, the two polypeptides were formed at equal or different rates. The relative rate of biosynthesis was constant for an individual cell line, suggesting that both allelic products were under separate genetic control. In a group of 21 unrelated individuals, the gene frequency of alleles for the 62- and 59.5-kDa precursor forms was 3:1. The two allelic forms of the arylsulfatase A polypeptides were converted into a 57-kDa form by endo-beta-N-acetylglucosaminidase H, an enzyme specifically removing asparagine-linked oligosaccharides of the high-mannose (and hybrid) type. The apparent difference in the number of asparagine-linked oligosaccharides suggests that the two allelic genes differ in a region coding the sequence Asn-X-Thr(Ser), which is required for attachment of asparagine-linked oligosaccharides.

Characterization and differential expression of an endothelial cell-specific surface antigen in continuous and sinusoidal endothelial, in skin vascular lesions and in vitro.

Continuous and sinusoidal endothelial cells display marked morphological and functional heterogeneity as to their plasmalemmal vesicle content, to the kind of intercellular junctional complexes, to the existence and kind of fenestrae and gaps, to the existence and character of their basement membrane, to their ability for phagocytosis and to other functional parameters. Monoclonal antibody 1F10, raised against human umbilical vein endothelial cells (HUVE cells), reflects these differences in recognizing--without any nonendothelial side reactions--an endothelial cell surface antigen, abundantly expressed in continuous endothelia, low and inconsistently expressed in liver sinusoidal and dermal lymphatic endothelia and absent from splenic sinusoidal endothelial cells. In differentiated skin vascular tumors, 1F10 antigen is expressed in normal amounts while it is only low and inconsistently expressed in the dedifferentiated endothelial cells of Kaposi's sarcoma and hemangiosarcoma. HUVE cells in culture, in contrast to their in situ ancestors, express variable amounts of 1F10 antigen. When endothelial cell-conditioned medium (ECC medium) is supplied to HUVE cells in culture, no 1F10 antigen is expressed, while supplementation with fresh serum-containing medium (FSC medium) or cytokines, such as bFGF, suffices to maintain 1F10 expression in 10-70% of the cells. From this we conclude that developmental regulation, environmental influences and cytokine supply contribute to the differentiation and maintenance of the 1F10+ and 1F10-endothelial cell phenotypes, both in vivo and in vitro.

Biosynthesis and maturation of alpha-N-acetylglucosaminidase in normal and Sanfilippo B-fibroblasts.

The biosynthesis of alpha-N-acetylglucosaminidase in normal and Sanfilippo B fibroblasts was studied by labeling cells with [35S]methionine and isolation of the enzyme by immunoprecipitation. The immunoprecipitated polypeptides were separated by polyacrylamide gel electrophoresis and visualized by fluorography. alpha-N-acetylglucosaminidase is synthesized as a precursor of an apparent mol. wt. of 87,000. Intracellular processing of the precursor yields two polypeptides of apparent mol. wts. of 73,000 and 76,000 via several intermediates. It is accomplished within 3 days after synthesis. Less than 30% of the newly synthesized precursor is secreted. In the presence of 10 mM NH4Cl, secretion is enhanced to more than 80%. In our study, no alpha-N-acetylglucosaminidase polypeptides could be detected in fibroblasts from patients affected with either the severe or mild form of Sanfilippo disease, type B.

Heterogeneity in late-onset metachromatic leukodystrophy. Effect of inhibitors of cysteine proteinases.

The synthesis of arylsulfatase A polypeptides was followed in fibroblasts from 11 patients with late-onset forms of metachromatic leukodystrophy. In 10 cell lines, the apparent rate of synthesis was 20%-70% as measured by the amount of [35S]arylsulfatase A secreted in the presence of 10 mM NH4Cl. The specific activity of the secreted arylsulfatase A was normal. The residual activity of arylsulfatase A was below 10% except for one cell line in which it was 20%. The activity of arylsulfatase A and the degradation of sulfatides was partially restored in these fibroblast lines by treatment with irreversible (peptidyl diazomethyl ketones) or competitive (leupeptin) inhibitors of cysteine proteinases. Thus, the mutation(s) in these cell lines led to the synthesis of arylsulfatase. A polypeptides with increased susceptibility to cysteine proteinases. Multiple allelic mutations within this group of late-onset metachromatic leukodystrophy were suggested by the clinical heterogeneity, the variability of the residual activity, and in the response to inhibitors of cysteine proteinases. In fibroblasts from one patient, the apparent rate of synthesis of arylsulfatase A was less than 5%. Furthermore, inhibitors of cysteine proteinases were without effect, suggesting that the mutation in this patient is different from the others.