Hideo Yaoita

Phagocytosis and bactericidal action of mouse peritoneal macrophages treated with leukotriene B4

The effects of exogenous leukotriene B4 (LTB4) on the resistance of mouse peritoneal macrophages against Salmonella (S.) typhimurium and Pseudomonas (P.) aeruginosa infections were studied. In vitro, LTB4 added to macrophage monolayers at final concentrations of 10(-12)-10(-8) M, enhanced their phagocytosis of S. typhimurium to 2.3 times the control level and that of P. aeruginosa to 1.8 times the control level. The intracellular killing rates were also elevated by the addition of LTB4: for S. typhimurium, 83.3% (LTB4) vs 59.1% (control) and for P. aeruginosa, 46.5% (LTB4) vs 9.2% (control). In vivo, intraperitoneally injected LTB4 (5 ng) enhanced the clearance at 24 h of intraperitoneally injected S. typhimurium from the mouse peritoneal cavity (2.38 x 10(3) +/- 0.94 x 10(3) cells [LTB4] vs 5.73 x 10(5) +/- 1.90 x 10(5) [control]) and spleen (5.00 x 10(2) +/- 0.94 x 10(2) [LTB4] vs 2.47 x 10(4) +/- 0.84 x 10(4) [control]), but this effect disappeared by 48 h. In contrast, in beige mice, an experimental model of the Chédiak-Higashi syndrome that is characterized by susceptibility to bacterial infection, there was no induction of the eliminating effect by intraperitoneal injection of LTB4. Activation of macrophages by exogenous LTB4 seemed to have contributed to such an augmented resistance of macrophages to bacterial infection. This study suggested a possible use of LTB4 in bacterial infectious diseases whereby phagocytes are able to play a key role in host defense.

Phorbol ester- and calcium-induced reorganization of 180-kDa bullous pemphigoid antigen on the ventral surface of cultured human keratinocytes as studied by immunofluorescence and immunoelectron microscopy

The hemidesmosome is an adhesion structure of the epidermal-dermal junction in keratinocytes. When keratinocytes migrate laterally or upward to differentiate, they must control the formation and disintegration of the hemidesmosomes. When keratinocytes are cultured in low-calcium (below 0.1 mM) medium, all cells behave like basal cells, adhere to the culture dish, and proliferate without differentiation. The calcium addition induces the differentiation. A bullous pemphigoid antigen, 180-kDa BPA, has been shown to be a component of the hemidesmosome. Using a monoclonal antibody to the 180-kDa BPA and a human squamous cell carcinoma cell line (DJM-1 cells), the fate of hemidesmosomes was studied after the addition of calcium to low-calcium-grown cells and 12-tetradecanoylphorbol-13-acetate (TPA) to high-calcium (1.87 mM) grown cells by immunofluorescence and immunoelectron microscopy. The antigen was distributed evenly as fine dots on the entire ventral surface of low-calcium cells, whereas they formed a peculiar, concentric ring or arch arrangement on the ventral surface of high-calcium cells. Immunoelectron microscopy revealed the deposits of gold particles at sites on the membrane surface, where some filamentous or electron-dense materials were associated, although the complete structure of hemidesmosomes was not formed. They deposited directly onto the membrane surface in low-calcium cells and with a distance of 20-50 nm from the membrane surface in high-calcium cells. The calcium addition caused a profound reduction of the 180-kDa BPA-positive area for 30 to 120 min and then formed the high-calcium-ring pattern after 4 to 6 h. A similar calcium response was seen in normal human keratinocytes. TPA (16 nM) treatment caused disintegration of the ring pattern in high-calcium DJM-1 cells. This was inhibited with a protein kinase C (PKC) inhibitor. H7 (20 microM). These results suggest that the hemidesmosome is a dynamic structure and PKC can be one of the major factors in controlling the hemidesmosome, since it is known that the low-high calcium shift induces a calcium influx and a PKC activation, and TPA activates PKC in keratinocytes.

EPIDERMOLYSIS BULLOSA ACQUISITA DIAGNOSED BY IMMUNOELECTRON MICROSCOPY

A 60‐year‐old Japanese man with a five year history of bullous lesions associated with scar and milia is described. This case seemed to have an unusual type of bullous pemphigoid clinically, histologically and immunofluorescently, but was found to have epidermolysis bullosa acquisita (EBA) based on electron microscopic and immunoelectron microscopic findings, in which amorphous substance and immunological reaction products were detected mainly below the subbasal lamina‐anchoring fibril zone.

A CASE OF GRANULOMA ANNULARE AND SARCOIDOSIS

Granuloma annulare (GA) and sarcoidosis are two diseases of unknown causes with epithelioid cell granuloma formation. In this paper, we discuss a patient with manifestations of GA and both systemic and cutaneous sarcoidosis. We examined serum MIF and serum MAF to study the relationship between GA and sarcoidosis. Only the serum from a patient in the active stage of sarcoidosis had significant MIF activity. Additional significant MAF activity was found in the active stages of sarcoidosis and GA, especially the stage of developing GA. It may be characteristic that GA has a high potential MAF activity.

Induction of macrophage disappearance reaction by immunoadsorbed MIF

Abstract The macrophage disappearance reaction (MDR) was induced by intraperitoneal (ip) injection of the immunoadsorbed macrophage migration inhibitory factor (MIF) fraction into the guinea pigs with macrophage-rich peritoneal exudates. The MDR induced by the immunoadsorbed MIF fraction was suppressed by α- l -fucose. Heparin, which is an anticoagulant drug, could also inhibit the MDR induced by the immunoadsorbed MIF fraction. Immunofluorescent study using fluorescein isothiocyanate (FITC)-labeled rabbit anti-human fibrinogen revealed the presence of fibrin/fibrinogen as an enveloping network pattern on the surface of macrophages during the course of MDR induced by the immunoadsorbed MIF fraction.

Control of the Distribution of Hemidesmosome Components in Cultured Keratinocytes: Ca2+ and Phorbol Esters

DJM‐1 cells (a human squamous cell carcinoma cell line) grown in low Ca2+ medium did not form cell‐cell junctions of desmosome‐keratin intermediate filament (KIF). When they were shifted to normal (high) Ca2+ medium, rapid translocation of desmoplakins from the cytosol to the plasma membrane to form desmosomes and reorganization of 180 kd‐hemidesmosome proteins were induced almost simultaneously. In correlation with these morphological responses, the Ca2+ shift caused a breakdown of inositol phospholipids, a formation of diacylgycerol (DAG) and inositol trisphosphate (IP3), protein kinase C (PKC) activation, and Ca2+ influx. 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA)‐treatment of low Ca2+‐grown DJM‐1 cells also caused desmosome formation in association with PKC activation. These TPA effects were cancelled with PKC inhibitors, 1‐(5‐isoqunolinylsulfomyl)‐2‐methylpiperazine (H7) and staurosporine. Treatment with other PKC‐activating agents, phorbol‐12,13‐butyrate (PDBu) and diaoctanoylglycerol (DOG), also induced desmosome formation. TPA‐treatment of normal Ca2+‐grown cells collapsed the organized distribution of the 180 kd‐hemidesmosome protein and appeared to detach this protein from the cell‐matrix adhering sites. This effect was also inhibited by H7. These results suggest that PKC activation plays important roles in upregulation of cell‐cell junctions and downregulation of cell‐matrix junctions in association with differentiation of keratinocytes.

An inhibitory factor against monocyte spreading in the sera of patients with systemic lupus erythematosus.

The effect of the sera of patients with systemic lupus erythematosus (SUE) on monocyte function was studied using cell spreading as an indicator. Monocyte spreading induced by exogenous stimuli was shown to be inhibited by SLE sera. Gel filtration of SLE sera on Sephadex G‐200 revealed that the factor responsible for this inhibition had a molecular weight of about 50,000. Pretreatment of monocytes with the inhibitory factor led to suppression of cell spreading induced by subsequent stimulation, but this hyporeactivity was reversible. Spreading of monocytes was rapidly aborted by the addition of this inhibitory factor. Thus, the inhibitory factor appeared to affect monocyte itself, but its effect seemed to be transient.

Serum Lipid Peroxide Levels in Various Dermatoses

In studies of physiological roles of lipid peroxide in cutaneous tissue, we examined serum lipid peroxide levels in 199 patients with various dermatoses such as psoriasis, eczema/prurigo, alopecia, bullous disorders, acne/seborrheic dermatitis, atopic dermatitis, SLE, urticaria, progressive systemic sclerosis, generalized morphea, and herpes zoster, by using the TBA (thiobarbituric acid) method and a new assay technique, called the MCDP (methyl carbamoyl‐dimethylamino phenothiazine)‐Hb method. The following results were obtained. By the TBA method, statistically significantly high serum lipid peroxide levels were noted in patients with psoriasis, eczema/prurigo, alopecia, SLE and generalized morphea. By the MCDP‐Hb method, similarly high levels were found in patients with alopecia and atopic dermatitis, compared with those of the control group. The discrepancy between the results from the TBA and the MCDP‐Hb methods is thought to be due to the fact that TBA method measures a secondary product of lipid peroxide, malondialdehyde, whereas the MCDP‐Hb method measures lipid hydroperoxide itself.

Regulatory mechanisms of cutaneous delayed-type hypersensitivity: II. Suppression of cutaneous delayed-type hypersensitivity by macrophage disappearance reaction

Studies were performed on the behavior of cutaneous delayed-type hypersensitivity (DTH) in guinea pigs in which macrophage disappearance reaction (MDR) was induced. Guinea pigs were immunized with dinitrophenylated egg albumin (DNP-EA), followed by intraperitoneal (ip) injection of liquid paraffin in order to elicit peritoneal macrophages. Subsequently 20 micrograms of EA was injected into these animals and the animals were divided into two groups. One group of animals was sacrificed for estimation of MDR 6 hr after the subsequent ip injection. The other group received a skin test by EA at the time of the subsequent ip injection. The first group of animals sacrificed for estimation of MDR exhibited a marked reduction in the number of peritoneal macrophages. The second group of animals that received skin tests revealed suppressed skin reactions 24 hr after the subsequent ip injection. A similar experiment was performed using the guinea pigs doubly immunized with DNP-EA and dinitrophenylated bovine gamma-globulin (DNP-BGG). Induction of MDR was performed by ip injection of BGG and skin tests were done by both EA and BGG. As a result, suppression of not only BGG-induced skin reactions but also EA-induced skin reactions was observed in animals in which MDR had been induced by BGG. In addition, the guinea pigs in which MDR was induced showed hyporeactivity to phytohemagglutinin (PHA). Reactivity to skin reactive factor (SRF) was also suppressed in these animals. The culture supernatants of macrophages incubated with the MIF fraction in vitro showed the ability to suppress skin reactions of cutaneous DTH, PHA and SRF.

Regulatory mechanisms of cutaneous delayed-type hypersensitivity. I: Suppression of cutaneous delayed-type hypersensitivity by migration inhibitory factor

The macrophage migration inhibitory factor (MIF) fraction was prepared from the immunoadsorbent column by using anti-guinea pig MIF antiserum. Suppression of cutaneous delayed-type hypersensitivity was achieved by intraperitoneal injection of the MIF fraction into the animals bearing macrophage-rich peritoneal exudates. Skin reactions induced by phytohemagglutinin (PHA) were also suppressed in these animals. Reactivity to skin reactive factor (SRF) was suppressed in these animals as well. The sera obtained from these animals exhibited the inhibitory activity against production of lymphokines from sensitized lymphocytes.