Leukocyte migration inhibition factor in atopic dermatitis: induction by concanavalin A in vitro.

Assays for the production of leukocyte migration inhibition factor (LMIF) activity by cultured lymphocytes in the presence of the T-cell mitogen concanavalin A (ConA) have been shown to be highly sensitive for detecting decreases in cell-mediated immunity in patients with malignant diseases and primary and secondary immunodeficiency diseases. We have applied the leukocyte migration inhibition test in agarose using supernatants from ConA-stimulated lymphocyte cultures (indirect leukocyte migration inhibition test in agarose for studying patients with atopic dermatitis. Only 5 of 14 lymphocyte cultures from atopic dermatitis patients produced LMIF when stimulated with ConA, as compared with 34 of 34 controls. This difference is highly significant.

Clinical study of a patient with lupus vulgaris before and after injection of dialyzable transfer factor.

This report describes the clinical improvement and acquisition of tuberculin skin-test sensitivity by a tuberculin-negative, drug-resistant patient with lupus vulgaris after a single injection of dialyzable transfer factor (TFd) from a tuberculin-positive healthy donor. The patient's lymphocytes showed a slight response to tuberculin in the leukocyte migration inhibition test and in the lymphocyte transformation test before TFd injection. The acquisition of cellular immunity to tuberculin was demonstrated in vitro by enhanced tuberculin-induced blast transformation. A good correlation between skin test and in vitro tuberculin sensitivity and clinical improvement was seen during the three years that the patient was observed.

Focal dermal-epidermal separation and fibronectin cleavage in basement membrane by human mast cell tryptase.

Mast cell proteases are believed to participate in the basement membrane destruction in blistering diseases. Thus, normal human skin specimens were incubated with purified human skin tryptase or compound 48/80 (a mast cell degranulator) for up to 24 h. Thereafter, the specimens were studied immunohistochemically. Tryptase caused, in the presence and absence of 1,10-phenanthroline, focal dermal-epidermal separation above laminin and almost complete disappearance of the staining of the extra domain A region of cellular fibronectin in and beneath the basement membrane. The immunopositivity of the cell-binding region of fibronectin, laminin, and collagens IV and VII, however, was unaltered. Compound 48/80 induced almost complete dermal-epidermal separation above intact laminin and only focal reduction in the extra domain A region of cellular fibronectin staining. These alterations by compound 48/80 were prevented partially by Nalpha-p-tosyl-L-lysine chloromethyl ketone or 1,10-phenanthroline alone but completely when both inhibitors were present suggesting the involvement of tryptic serine proteinases, probably also tryptase, and metalloproteinases. Preventive effect of N-tosyl-L-phenylalanine chloromethyl ketone was weak suggesting minor function of chymotryptic serine proteinases. When tryptase was incubated with heparin and pure plasma fibronectin, an abrupt decrease in the adherence of cultured keratinocytes on to plastic surface coated with these substances and a gradual plasma fibronectin cleavage to 173, 161, and 28 kDa fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis were found. In conclusion, tryptase can cause focal dermal-epidermal separation above laminin in skin specimens but it is not known to what extent the decreased keratinocyte adherence in vitro and fibronectin cleavage are related to this dermal-epidermal separation.

Immunologic features of chronic granulomatous mucocutaneous candidiasis before and after treatment with transfer factor.

We report the acquisition of skin test sensitivity to Candida albicans antigen and the ability to produce leukocyte migration inhibition factor (MIF) by a Candida-negative patient with chronic granulomatous mucocutaneous candidiasis after treatment with dialyzable transfer factor (TFd). The TFd was acquired from Candida-positive healthy donors. Three of seven attempts to transfer Candida skin test sensitivity were successful, and the acquired skin reactivity lasted for 12 to 21 days. The acquisition of cellular immunity to Candida was demonstrated in vitro by production of leukocyte MIF. No Candida-induced lymphocyte transformation was observed before or after TFd injection. The TFd did not cause Candida-induced blast transformation when added directly to cultures of lymphocytes from the patient. Pain, tenderness, redness, and edema were observed around the Candida granulomas on each occasion when the skin test to Candida became positive. Two weeks after TDd injection, the proliferative response of peripheral blood lymphocytes increased, as measured by incorporation of tritiated thymidine into lymphocytes within the first hour of in vitro incubation.

Cell-mediated immunity in vitro in atopic dermatitis.

The function of T cells in atopic dermatitis was studied by leukocyte migration agarose and lymphocyte transformation tests. We found that phytohemagglutinin (PHA)- and PPD-induced release of leukocyte migration inhibition factor (LIF) from lymphocytes is significantly decreased (P less than .001) in patients with atopic dermatitis as compared with healthy controls. Blastogenic response of lymphocytes induced by PPD was also decreased in atopic patients as compared with controls (P less than .01). No differences were found in spontaneous blastogenesis or in blastogenic response of lymphocytes in vitro to PHA, concanavalin A (ConA), or streptokinase-streptodornase (SK-SD) between patients with atopic dermatitis and controls.

Effect of drugs on histamine radio-enzyme assay.

The effect of over 200 drugs and other compounds on histamine radio-enzyme assay was studied. Some muscle relaxants (e.g. alcuronium), some sympathomimetics (e.g. dopamine, isoxsuprine, tyramine and possibly phenylethylamine), antimalarial drugs, procaine, procainamide, Berenil and serotonin were potent compounds to interfere with this assay. In some special cases still potentially inhibitory drugs seemed to be some muscle relaxants (e.g. vecuronium, pancuronium and tubocurarine), antidepressants, antihistamines (e.g. cimetidine, ranitidine and diphenhydramine), chinidin, disopyramide, tolazoline and salazosulfapyridine.

Structure and function of salivary glands in psoriatics.

A total of 28 psoriatics and the same number of age-and sex-matched healthy individuals as controls were subjected to clinical, histopathologic, and salivary flow rate studies to assess whether structural and functional disturbances attributable to psoriasis vulgaris are detectable in their salivary glands, i.e., whether they have sicca syndrome or not. The oral status of the patients in the two series proved to be normal, and no clinical signs of impaired tear and salivary secretion were notable, as determined by Schirmer test I and stimulated parotid flow rate measurement, respectively. In labial biopsy, the degree of salivary gland inflammation, fatty infiltration, and fibrosis were equal in both series. These results, seemingly contradictory to those of earlier workers, are discussed in the light of the newly introduced concept on MALT (mucosal associated lymphatic tissue), and the conclusion is drawn that salivary glands are not affected by psoriasis vulgaris nor complicated by arthritis. The latter seems to be required to initiate the inflammatory reaction within MALT, in which synovial tissue and salivary glands are included. The necessity of an age- and sex-matched control series is emphasized whenever salivary gland changes in association with systemic diseases are studied.

The development of manifest psoriatic lesions is linked with the appearance of ICAM-1 positivity on keratinocytes.

The development of psoriatic lesions was studied in 36 psoriatic patients using the Koebner reaction induced by tape stripping. Two biopsies per patient were taken from non-lesional psoriatic skin before, and 6 h, 2 days, 7 days, 14 days and 21 days after tape stripping. Alterations in HLA-DR, ICAM-1, Ki-67 and FXIIIa positivities in both the dermis and the epidermis were estimated using immunohistochemical methods. A double staining for CD4+ and CD8+ T cells was also carried out to show their possible Ki-67 positivity. Results were compared with those from lesional (mature plaque) and non-lesional psoriatic skin, and control skin. Of the 36 patients, 9 were Koebner-positive. The most important finding in Koebner-positive psoriatic skin was the appearance of ICAM-1 positivity on epidermal keratinocytes simultaneously with the clinically observed lesion on day 7. The number of FXIIIa+ dendrocytes in the dermis was quite constant, and increased in mature psoriatic lesions only. The number of active HLA-DR+ immunocompetent cells increased in developing psoriatic lesions, being highest in mature lesions, but no Ki-67 positivity was detected in epidermal or dermal T cells in the psoriatic specimens. Based on these results, it is concluded that T cells divide and are activated extracutaneously in psoriasis, and also that ICAM-1/LFA-1 interactions are important in the recruitment of inflammatory cells and in controlling the effector cell functions.

The development of manifest psoriatic lesions is linked with the invasion of CD8 + T cells and CD11c + macrophages into the epidermis.

Koebner response was studied in 35 psoriatic patients. Two punch biopsies per patient were taken from non-lesional psoriatic skin before, and 6 h, 2 days, 7 days, 14 days and 21 days after, tape stripping. Alterations in the numbers of CD1+ Langerhans cells, CD4+ and CD8+ T cells and CD11c+ macrophages were mapped morphometrically. Results were compared with lesional and non-lesional psoriatic skin, and control skin. Nine of 35 patients were Koebner-positive. No statistically significant differences were noted between non-lesional psoriatic and control skin. CD4+ T cells increased in number 2 days after trauma in both the epidermis and the dermis, whereas epidermal CD8+ T cells and CD11c+ macrophages increased only in the Koebner-positive lesional skin after 7 days. The changes in lesions induced by tape-stripping resembled those seen in lesional psoriatic skin (mature plaques). The number of CD1+ cells increased in mature psoriatic lesions only. It seems possible that trauma per se stimulates the accumulation of CD4+ T cells at the site of injury, but the development of manifest psoriatic lesions correlates with invasion of CD8+ T cells and CD11c+ macrophages into the epidermis.

Immunohistochemical analysis of sensory nerves and neuropeptides, and their contacts with mast cells in developing and mature psoriatic lesions.

The distribution of the neuropeptides substance P (SP), vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) was studied immunohistochemically in psoriatic skin during the Koebner response (6 h, 2 days, 7 days, 14 days, 21 days), and in mature psoriatic plaques, of 37 psoriatic patients. The morphological association of sensory nerves, SP and VIP with papillary mast cells was also monitored. The nerves containing SP, VIP or CGRP were very scanty in control skin, and in non-lesional and Koebner-negative psoriatic skin. The first psoriatic lesions were seen 7 days after tape stripping the symptomless psoriatic skin. SP- and VIP-containing nerves were slightly increased in Koebner-positive specimens, but the increase was very prominent in dermal papillae of mature psoriatic plaques. In the plaques, nerve-mast cell contacts were significantly increased (p < 0.001) compared with non-lesional psoriatic skin. Only SP-positive fibres were detected in the epidermis and in contact with papillary mast cells. VIP was mainly located around capillaries where SP was also found. No change was noted in CGRP-positive fibres between lesional and non-lesional specimens. The appearance of SP and VIP in the capillary walls is morphological evidence for their function as vasodilators in psoriatic lesion. A slight increase in SP- and VIP-positive fibres in Koebner-positive specimens suggests that these neuropeptides may participate in the inflammatory reaction at an early stage. Their prominence in mature psoriatic plaques in turn indicates a role for them in the maintenance of psoriatic lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme- and immunohistochemical localization of mast cell tryptase in psoriatic skin.

The localization of mast cell tryptase in involved and noninvolved skin sections from 12 psoriatic patients was investigated using both enzyme- and immunohistochemical staining techniques. Each involved skin section contained an increased number of tryptase-positive mast cells in the superficial dermis as compared with corresponding noninvolved skin sections. The substrate-hydrolyzing and inhibition properties for tryptase activity in involved and non-involved psoriatic skin sections were identical with each other as well as with those previously described in normal human skin or mastocytoma skin sections. In four patients, epidermal enzyme activity was observed, but only in the involved skin. None of the uninvolved sections showed tryptase activity in the epidermis. This activity was not inhibited by alpha 1-antitrypsin, and after removing the enzyme-histochemical stain with Tween 20, positive results obtained with tryptase-specific antibody were found in the same locations. In addition, tryptase-positive cells were detected in close contact to lesional epidermis, but without epidermal staining in four patients. In the epidermis, the positive staining was granular, and active tryptase was detected as far as the stratum corneum. This study is the first description of the presence of active mast cell tryptase in psoriatic epidermis, where this enzyme may have a role in increased cell division.

Mast cell proteinases and cytokines in skin inflammation.

The role of mast cells in provoking immediate-type hypersensitivity reactions is well established, but their involvement in chronic inflammation and immune reactions is not so clear. Mast cells synthesize and secrete large amounts of active proteinases, including tryptase, chymase, carboxypeptidase and cathepsin G, which can rapidly process numerous biologically active peptides and proteins or their precursors. Furthermore, mast cells are able to produce a variety of cytokines such as interleukin-4 (IL-4), IL-5, IL-6, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) which are known to be intensively involved in modulating and directing inflammatory responses in the skin. In this review, the role of mast cell proteinases and cytokines in skin inflammation is discussed.

Hyaluronan and CD44 in psoriatic skin. Intense staining for hyaluronan on dermal capillary loops and reduced expression of CD44 and hyaluronan in keratinocyte-leukocyte interfaces.

The distributions of hyaluronan (HA) and its presumptive receptor, CD44, were studied in skin samples from 13 psoriasis vulgaris patients, using an HA-specific probe (HABC), and monoclonal antibodies, respectively. The general distribution of HA and CD44 in psoriatic lesional epidermis resembled that in normal epidermis. However, areas of epidermis invaded by leukocytes showed a local depletion of HA and CD44, particularly at the contact areas of keratinocytes to lymphocytes and neutrophils. Removal by cellular uptake or extracellular degradation of CD44 and HA may be required for tight adherence between a keratinocyte and a leukocyte. On the dermal side, the tips of the prolonged dermal papillae in psoriatic lesions were intensively stained with HABC. The dilated capillaries and the space below the tip basal lamina, in particular, were heavily covered with HA. Occasionally, a similar intense staining was seen around an enlarged capillary in uninvolved psoriatic skin. CD44-positive leukocytes were found around the affected capillaries. The accumulation of HA in the dermal papillae may support the growth of psoriatic lesions, since HA stimulates the growth of capillaries as well as attracting inflammatory cells.

Quantitative analysis of contact sites between mast cells and sensory nerves in cutaneous psoriasis and lichen planus based on a histochemical double staining technique.

The aim of the present study was to test further our previous hypothesis that the inflammatory reaction in psoriasis is neurogenic. For this purpose, contact sites between mast cells and sensory nerves were morphometrically analysed in the basement membrane zone, papillary dermis and three dermal zones of lesional/non-lesional psoriatic and lichen planus skin as well as in healthy control skin. The analyses were made on sections stained with a histochemical double stain developed for this study. With the double stain, active mast cell tryptase was stained blue enzyme histochemically, and the sensory nerves black using specific monoclonal anti-neurofilament antibodies with immunogold. In psoriatic lesions, both mast cells and mast cell--nerve contacts were markedly more frequent in the basement membrane zone and in the papillary dermis when compared with the corresponding areas in the other groups. Mast cell numbers were increased in both lesional and symptom-free skin in lichen planus, but no increase was found in the mast cell--nerve contacts. Increased contacts between mast cells and sensory nerves indicate that the elements exist for neurogenic inflammation in psoriatic lesions. These increased contacts are not due to the extensive inflammatory reaction only, because they were not observed in lichen planus lesions.

Quantitative digital image analysis applied to demonstrate the stratified distribution of involucrin in organ cultured human skin.

In this study, quantitative digital image analysis was utilized to measure the optical density of immunostains of involucrin at different depths in the epidermis to obtain reliable ordinal-scaled interpretations of the staining intensity. The distribution of involucrin within the epidermis was investigated in air-liquid interface and submerged skin organ cultures at different time-points. A greyscale calibration procedure to standardize the optical units was used. By the 2nd day of culture, staining of involucrin had shifted markedly towards the mid or basal epidermis. Air-liquid interface cultures showed a less intensive shift than the submerged cultures. Up to the 7th day, involucrin staining remained in the upper epidermis in the air-liquid interface cultures, though weak staining was already observed in the basal epidermis. The results suggest that air-liquid interface conditions maintained physiological conditions better than submerged conditions which result in cultures that may have to increase their involucrin synthesis to improve the barrier function against the surrounding liquid during culture. Alternatively, changes in involucrin synthesis could reflect disturbed homeostasis. Concentrating measurements on certain cell layers might give more detailed information about changes in involucrin expression. Although the detection method was used to study the histochemistry of skin, it could easily be applied to other tissues as well.

Release of histamine and leukotriene C4 in immediate allergic wheal reaction as measured with the microdialysis technique.

Other mediators as well as histamine can contribute to the allergic wheal reaction. In this study, the microdialysis technique was used to monitor the release of histamine, leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) in prick-test wheal reactions induced by cow dander allergen. Of 31 atopic subjects, 25 showed detectable histamine release that correlated significantly with the number of tryptase-positive mast cells and serum cow-specific IgE but not with the wheal size. Detectable LTC4 release was shown by 16 of 18 subjects, but PGD2 release was shown by only 7 of 17 subjects, and neither mediator was associated with tryptase-positive mast cells, IgE levels or wheal size. An inverse association between histamine release and LTC4 release in these 18 subjects was found rather than a direct correlation. With advancing age of the subject histamine release (n = 31) tended to decrease, although insignificantly, but LTC4 release (n = 18) and sensitivity to histamine prick increased significantly, which seemed to parallel the changes in the wheal size induced by cow allergen. In conclusion, the results showed that the release of histamine, LTC4 or PGD2 alone cannot explain the extent of the wheal reaction. In addition, the amount of histamine released was not related to the amount of LTC4 released, but rather an inverse association existed between these mediators.

Quantitative enzyme-histochemical analysis of tryptase- and chymase-containing mast cells in psoriatic skin.

Tryptase-containing mast cells have recently been found to be increased in the upper dermis of psoriatic lesions. In the present study, the distribution of chymase- and tryptase-containing mast cells was morphometrically analysed at different dermal levels of lesional and non-lesional psoriatic skin (12 patients) as well as normal human skin. Mast cell tryptase was identified enzyme-histochemically, using Z-Gly-Pro-Arg-MNA as the substrate. For demonstrating mast cell chymase, a simple and specific enzyme-histochemical staining method was developed, using Suc-Val-Pro-Phe-MNA as the substrate. All mast cells positive for chymase were also positive for tryptase and Giemsa stain. Although the number of tryptase-positive mast cells was slightly increased throughout the dermis of lesional psoriatic skin, this increase was most pronounced in the upper dermis immediately beneath, and in close contact with, the epidermis. In contrast, the number of chymase-positive mast cells was clearly decreased in the upper dermis of psoriatic lesions, but not in the deeper dermis, as compared with non-lesional psoriatic skin. In addition, all chymase-positive mast cells observed in the upper dermis were very weakly stained when compared with those in the deeper dermis. No differences were found between non-lesional psoriatic skin and normal skin in which the number of mast cells containing chymase was 72-73% of the number containing tryptase. The present results suggest that T mast cells particularly, containing tryptase but no chymase, proliferate in psoriatic lesions, and that the increase in tryptase activity and the decrease in chymase activity in the upper dermis may lead to an imbalance in the biochemical regulatory systems.

Biochemical and histochemical evaluation of tryptase in various human tissues.

The distribution of tryptase in various human tissue high-salt extracts (skin, lung, pancreas, liver, kidney, and spleen) was studied. Tryptase activity was compared with tissue histamine concentration, chymase activity, and cathepsin D, and histamine-N-methyltransferase (HMT) activities. Tryptase activity, found biochemically in tissue extracts, was localized in tissue sections by an enzyme-histochemical method using peptide 4-methoxy-2-naphthylamide substrates and Fast Garnet GBC as the chromogen. The highest levels of tryptase activity were found in lung and skin extracts. Liver, kidney, and spleen extracts displayed only a little activity. The distribution of histamine was similar to that of tryptase, whereas distributions of cathepsin D and HMT were quite different from that of tryptase. High-salt extracts of lung contained no detectable chymase activity, but in skin extracts this activity was high. Using an enzyme-histochemical method, the tryptase activity in tissue sections seemed solely to be confined to cells, which were granular and Giemsa positive after the red azo dye had been removed with Tween 20. Skin and lung sections contained the highest number of positively stained cells. The inhibition properties of tryptase, found in both tissue extracts and sections, and the substrate profile in tissue sections were identical. Human leukocyte preparation was negative for tryptase when stained enzyme-histochemically. The present results suggest that tryptase in human tissues is found only in the mast cells. The enzyme seems to be identical in the various human tissues studied because the different high-salt extracts were immunologically cross-reactive when tested with a rabbit polyclonal antibody against skin tryptase.

Mast cell tryptase and chymase in developing and mature psoriatic lesions.

The number and distribution of mast cells in non-lesional and lesional skin samples from 13 psoriatic patients were analyzed enzyme- and immunohistochemically. Mast cell tryptase was stained with the sensitive substrate Z-Gly-Pro-Arg-4-methoxy-2-naphthylamide, and chymase with Suc-Val-Pro-Phe-MNA and monoclonal B7 anti-chymase antibody. In addition, healthy-looking skin from 27 psoriatic patients was tape-stripped resulting in induction of the Köbner response in 9 patients. Sequential biopsies were taken before and after (7, 14 and 21 days) tape-stripping, and both tryptase and chymase were stained enzyme-histochemically. In non-lesional psoriatic skin, 70 +/- 24% (mean +/- SD) of the mast cells contained chymase enzyme activity, and 78 +/- 18% chymase immunoreactivity. About 10% of the chymase-immunoreactive cells lacked chymase activity. In lesional psoriatic skin, tryptase-positive cells were increased in number throughout the dermis but especially beneath the epidermis. Chymase immunoreactivity paralleled the tryptase activity, whereas chymase activity was strongly diminished both in terms of mast cell numbers and in staining intensity in the papillary dermis. The apparent inactivation of chymase may be due to the action of the chymase inhibitors, alpha 1-antitrypsin and alpha 1-antichymotrypsin, localized immunohistochemically in mast cells of lesional and non-lesional psoriatic skin. In the developing psoriatic lesion, mast cells displaying chymase activity were already 27-38% decreased in number in the upper dermis on day 7 after tape-stripping, along with the first clinical signs of psoriasis.(ABSTRACT TRUNCATED AT 250 WORDS)

Dermatoses determined in a population of farmers in a questionnaire-based clinical study including methodology validation.

OBJECTIVES: The study analyzed skin diseases in a population sample of Finnish farmers descriptively and in the process validated the question "Do you have a skin disease now?" METHODS: All farmers from one Finnish municipality were surveyed for dermatoses, first with a questionnaire and then with a clinical examination of those who reported dermatoses. Another population of farmers answered a set of questions immediately before a clinical examination, and the self-report of current dermatosis was validated. RESULTS: Eczema was diagnosed for 66% of the women and 53% of the men who had reported dermatosis in the questionnaire study 6 to 12 months earlier. Toe-web maceration, psoriasis, folliculitis, and acne were, after eczema, the most frequent diagnoses (in that order). In more than 50% of the cases, the location of clinically determined dermatoses corresponded with the skin disease areas reported 6 to 12 months earlier. In the validation study, everyone who reported a skin disease immediately before the clinical examination were found to have a skin disease. In addition 22% of those not reporting dermatosis were found to have a skin disease. Toe-web maceration was the most common dermatosis not reported by the farmers. CONCLUSIONS: Finnish farmers suffered from the same type of dermatoses as other populations. The prevalence of eczema and hand eczema was similar to that of other risk populations. A self-report of current dermatosis is probably a good indicator of the point prevalence of explicit skin diseases in populations.