Decreased monocyte shedding of the migration inhibitor soluble CD18 in alcoholic hepatitis.

OBJECTIVES: During alcoholic hepatitis (AH) monocytes traverse the vascular boundaries and massively invade the liver. In principle, tissue extravasation can be limited through shedding of CD18 integrins from leukocytes, including monocytes. The soluble (s) product sCD18 conceals adhesion receptors on the endothelium, which reduces monocyte extravasation. In AH, monocytes are dysfunctional, but whether this involves their self-generated anti-migration is unknown. Our aim was, therefore, to investigate monocyte CD18 dynamics in AH. METHODS: We studied 50 AH patients and 20 healthy controls. We measured monocyte expression and conformational activation of CD18, plasma (P)-sCD18, stimulated in vitro CD18 shedding and P-sCD18 in a short-term chronic-binge mouse model. RESULTS: AH-derived monocytes had a 30-60% higher expression of active CD18 receptors (p < 0.01), but the sCD18 concentration per monocyte was reduced in vivo by 30% and in vitro by 120% (p < 0.01). Ethanol reduced the in vitro shedding of CD18 in the patients only. TNFα increased sCD18 concentration per monocyte, but less so in the patients (p < 0.04). P-sCD18 per monocyte was inversely related to disease severity. In early alcoholic liver disease, P-sCD18 was decreased in the mouse model. CONCLUSIONS: The monocyte CD18 integrins are highly activated in AH and the single monocyte shedding of CD18 was decreased favoring tissue extravasation. Alcohol in itself and altered monocyte responsiveness to TNFα may explain this lowered shedding. TRANSLATIONAL IMPACT: The contribution of this mechanism to the excessive monocyte liver infiltration in AH should be further explored as it may serve as a potential therapeutic target to limit liver inflammation.

Directional migration of leukocytes: their pathological roles in inflammation and strategies for development of anti-inflammatory therapies.

Directional migration of leukocytes is indispensable to innate immunity for host defense. However, recruitment of leukocytes to a site of tissue injury also constitutes a leading cause for inflammatory responses. Mechanistically, it involves a cascade of cellular events precisely regulated by temporal and spatial presentation of a repertoire of molecules in the migrating leukocytes and their surroundings (microenvironments). Here I will summarize the emerging evidence that has shed lights on the underlying molecular mechanism for directional migration of leukocytes, which has guided the therapeutical development for innovative anti-inflammatory medicines.

Conditioned immunosuppressive effect of cyclophosphamide on delayed-type hypersensitivity response and a preliminary analysis of its mechanism.

In the present study, camphor odor and intraperitoneal (i.p.) injection of cyclophosphamide (CY) were used as conditioned stimulus (CS) and unconditioned stimulus (US), respectively. In the unconditioned group, mice were exposed to camphor odor for 1 h followed by an i.p. injection of CY (75 mg/kg). On the next day, the above CS/US association trial session was repeated followed by smearing dinitrochlorobenzene (DNCB) on mouse abdominal skin for sensitizing the animal for delayed-type hypersensitivity (DTH) response. Five days after DNCB sensitization, mice were exposed to camphor odor (1 h), followed by an i.p. injection of CY, and then DNCB was smeared on the left ear of mice for the challenge of DTH response. Both the left/right ear weight ratio and the activity of leukocyte migration inhibitory factor (LMIF) were used as the index of DTH response, which was done 24 h after DNCB challenge. In the conditioned group, the treatment was the same as that in the unconditioned group, except that normal saline was injected on day 5 instead of CY. Furthermore, in order to analyze the mechanism of the conditioned response (CR), the mouse serum from the conditioned group (CR serum) was injected into normal mice 6 h prior to DNCB challenge. Results showed that in the conditioned group, left/right ear weight ratio and LMIF activity were statistically lower than that in the DTH group, and there was no difference between conditioned and unconditioned groups. Thus, an animal model of conditioned immunosuppressive response had been established. The results also showed that after CR serum was injected into normal mice, DTH response was also significantly suppressed. However, if CR serum was treated with dialysis (10,000 molecular weight cut-off), the suppressive effect of CR serum on DTH response disappeared. Taken together, the data suggested that a chemical compound(s) in serum, with a molecular weight less than 10,000, was important in mediating the conditioned immunosuppressive response. This may be a very important molecule(s) that could be very critical to our understanding of the interaction between the central nervous system and immune function.

Leucocyte migration inhibition factor (L-MIF) in malnourished Nigerian children.

Leucocyte Migration Inhibition Factor (L-MIF) was measured in 41 children with marasmus, 19 with kwashiorkor, 5 with marasmic-kwashiorkor and 35 well-fed healthy children serving as controls. For L-MIF assay, two different antigens (live attenuated measles virus vaccine and diptheria pertussis tetanus (DPT) vaccine were used. Percentage migration indices obtained with the two antigens were significantly higher in the malnourished than in the well-fed healthy sex and age-matched controls (P < 0.01). The total serum protein and albumin concentrations were significantly reduced in the malnourished children compared with the controls (P < 0.01). Mean total leucocyte numbers were not significantly different in marasmic and marasmic-kwashiorkor children compared with the controls (P > 0.21).

Cell-mediated and humoral immunity in bulls infected with IBR/IPV virus (BHV 1).

Time-related changes in specific cell-mediated immunity (CMI) and in humoral immunity were monitored in 20 bulls, aging 12 to 16 months, in four groups, each consisting of 5 animals. Group I was experimentally and group III-naturally infected with BHV 1 virus, groups II and IV serving as control animals. Tests for CMI included delayed type hypersensitivity (DTH), leukocyte migration inhibitory factor (LMIF) and granulocyte migration inhibitor factor (GMIF-LIF-buffy coat leukocyte migration inhibitory factor) in the presence of BHV I antigen while humoral immunity was tested by serum induced neutralization of the virus (SN) and by estimation of serum IgG, IgG1, IgG2, IgM and IgA levels. Immunologic, virologic and clinical studies were performed two days before infection and 17 timed within 91 days after the infection in bulls of group I and II and 7 times within 42 days after developing the disease in bulls of groups III and IV. The results showed that positive CMI reactions appeared 3 to 4 weeks earlier than positive antibody titers in SN test. The antibodies belonged most probably to IgG1 and IgG2 subclasses.

[In vitro transfer of immunity against PPD with dialyzable extract of leukocytes from human colostrum]. / Transferencia in vitro de inmunidad a PPD con extracto dializable de leucocitos de calostro humano.

The aim of this study is to demonstrate the transference of PPD hypersensibility in an in vitro model, with dialysable colostral leukocyte extract (DCLE) of PPD+ and PPD-mothers, through measurements of leukocyte migration inhibition factor activity (LIF) from blood obtained of the umbilical cord of newborns from PPD+ mothers. The results show that DCLE PPD+ incubated with leukocytes of newborns from PPD- mothers had inhibition of leukocyte migration compared with migration of leukocytes incubated with DCLE PPD-. These results suggest that in this in vitro model, DCLE transfers hypersensibility to PPD.

Hypersensitivity of delayed type in hypertensive patients.

The agarose migration technique was used for demonstration of delayed-type hypersensitivity to arterial vessel wall antigens in patients suffering from chronic essential hypertension. By means of this technique, it was demonstrated that the migration indices from the hypertensive patients differed significantly from the normotensive control persons, P less than 0.005. The significant difference was abolished when anti-LIF was added to the migration tests. This means that a hypersensitivity of the delayed type had developed in the hypertensive patients and the results indicated that the hypersensitivity was an autoimmunity to arterial vessel wall antigens.

Cell mediated immunity in herpes simplex keratitis in man.

Varied manifestations of herpes simplex keratitis are postulated to be related to alterations or paucity of protective immune response to the virus. In this study lymphocyte subpopulations and macrophage inhibition factor (MIF) assay are investigated in herpes simplex keratitis. Active T-lymphocytes are detected to be significantly low in active keratitis as compared to the healed stage (P less than 0.001) and normal control subjects (P less than 0.01). Active T-cells are also lower in bilateral keratitis than in unilateral keratitis (P less than 0.01), and in stromal keratitis than in epithelial keratitis (P less than 0.05). Total E-rosette-forming cells and leucocyte migration inhibition factor (MIF) assay demonstrates significantly lower values in bilateral keratitis than in unilateral keratitis, and in stromal keratitis than in epithelial keratitis. On healing, total E-RFC, active T-cells and MIF values improved and are comparable to those found in normal subjects. Enumeration of absolute lymphocyte counts and EAC rosette-forming cells (B-lymphocyte cells) did not yield any differences. Our observations demonstrate paucity of cell-mediated immune response in stromal keratitis. More marked deficiency is demonstrable in bilateral keratitis. Manifestations of herpes simplex keratitis and their healing is observed to be relatable to the level of cell mediated immune response.

Applicability of the leukocyte migration inhibition test in the clinical practice.

The leukocyte migration inhibition test reveals in vitro the presence of lymphocyte sensitivity and, consequently, of cell-mediated immunity, to a given antigen. Applied in a variety of immune and allergic cases it proved to be useful for the positive diagnosis of the disease and/or for the detection of cell-mediated immune deficiency. The results obtained recommend the leukocyte migration inhibition test in the clinical practice.

Cellular immunity to nucleocapsid and pre-S determinants in asymptomatic carriers of hepatitis B virus.

Previous studies of cellular immunity in asymptomatic HBV carriers have been limited to evaluation of responses to plasma-derived HBsAg preparations. We have explored the specificity of cellular immune responses to HBV antigens in these subjects using an indirect T-lymphocyte migration inhibitory factor assay and three antigen preparations (recombinant nucleocapsid antigen (HBcAg), plasma-derived HBsAg with or without pre-S2, and Saccharomyces cerevisiae-synthesized HBsAg without pre-S2 region). T cells from 10 asymptomatic chronic HBV carriers with normal liver function tests were responsive to nucleocapside determinants (mean migration index = 0.55 +/- SD 0.07) and to pre-S2-positive plasma-derived HBsAg (MI = 0.62 +/- 0.05). However, none responded to HBsAg devoid of pre-S2 sequences (MI = 0.98 +/- 0.04). In further experiments, T cells from three HBV carriers, cultured with six different HBsAg preparations, exhibited responsiveness only to those preparations containing significant pre-S2 activities. Our results show that T-cell immunity to nucleocapsid determinants of the virus and HBsAg in present in asymptomatic HBV carriers; the latter is restricted to antigenic preparations containing significant pre-S2 activities. Hence, T-cell immunity to pre-S determinants may not always be associated with HBV clearance.

Cellular hypersensitivity to gluten derived peptides in coeliac disease.

Wheat gluten derived antigens have been tested for their ability to inhibit the migration of leucocytes from healthy subjects and patients with coeliac disease. Three preparations of a water soluble fraction (Frazer's fraction III, FIII) of partial peptic tryptic digests of wheat gluten had different effects in a direct (one stage) assay. Subfractions B and B2 caused migration inhibition of leucocytes from patients with treated coeliac disease but not of leucocytes from healthy volunteers or patients with Crohn's disease or ulcerative colitis. This migration inhibition seems to be specific for gluten fractions because maize zein fraction B, beta-lactoglobulin and ovalbumin did not cause it. The sensitivity of coeliac leucocytes to fraction B is not related to factors present in coeliac serum as the migration of leucocytes from healthy individuals preincubated with coeliac sera was not inhibited. Puromycin diminished inhibition by fraction B, which was active at 1.2 micrograms/ml in an indirect (two stage) migration inhibition assay; this is consistent with a process involving elaboration of lymphokine(s). More highly purified fractions of B2, P1-P4 were prepared by reverse phase high performance liquid chromatography (HPLC) and showed differing potency in direct and indirect assays, with P4 being the most active fraction. Inhibition of migration by gluten derived peptides appears to result from the release of lymphokine by leucocytes specifically from coeliac patients.

Mitogen induced LIF-production in T-lymphocyte subpopulations

El método de rosetas con eritrocitos de pollo recubiertos de IgG se ha utilizado junto con separación por flotación en ficoll-hypaque para obtener subpoblaciones de células enriquecidas o disminuidas de linfocitos T con receptor para Fc de IgG (Tg). Estas poblaciones celulars se han utilizado para determinar producción de LIF a diferentes concentraciones de los mitógenos, utilizando un método en microgota de agarosa. De esta manera, hemos podido observar una respuesta semejante en las diferentes poblaciones celulares hacia concanavalina A y una respuesta diferencial hacia fitohemaglitinina donde por lo general se observa una respuesta negativa de la población enriquecida con células Tg mientras que las poblaciones de linfocitos totales o aquellas en las que se han eliminado las Tg dan una respuesta positiva al segundo mitógeno

The effect of corticosteroids and other antineoplastic agents on the generation of leukocyte migration inhibition factor.

We have shown that hydrocortisone in physiological concentrations can inhibit the production of leukocyte migration inhibition factor (LMIF), but does not diminish the action of this lymphokine. Other agents tested failed to influence LMIF production. Inhibition of LMIF production by corticosteroids was influenced by the nature of the stimulus used for the production as an effect could be seen with PHA or Con A, but not Staphylococcal enterotoxin A (SEA). Production of LMIF was promptly restored after removal of the steroids. Furthermore, addition of a calcium ionophore to PHA restored the production of LMIF even in the presence of corticosteroids. In contrast, addition of exogenous IL-2 did not correct the defect in lymphokine secretion. We believe that inhibition of the production of LMIF by steroid may lead to defective granulocytic function and thus, predispose to infection.

[Effect of 3-hydroxyanthranilic acid-antigen on the migration of peripheral blood leukocytes in patients with bladder tumors]. / Vliianie 3-OAK-antigena na migratsiiu leikotsitov perifericheskoi krovi bol'nykh s opukholiami mochevogo puzyria.

The effect of 3-hydroxyanthranilic acid-antigen on peripheral blood leukocyte migration inhibition was studied in 75 patients with bladder malignancies. The antigen appeared to modify the said migration. A correlation was established between stage of tumor progression and the effect of 3-hydroxyanthranilic acid-antigen on leukocyte migration.

T-lymphocyte sensitization to hepatocyte antigens in autoimmune chronic active hepatitis and primary biliary cirrhosis. Evidence for different underlying mechanisms and different antigenic determinants as targets.

Cultured with a liver-derived lipoprotein complex, T lymphocytes from 42 of 45 patients with autoimmune chronic active hepatitis generated migration inhibitory factor compared with 16 of 33 patients with primary biliary cirrhosis. Unlike T lymphocytes from patients with primary biliary cirrhosis, the T-cell reactivity of patients with chronic active hepatitis was always suppressed by T cells from normal subjects and, with two exceptions, by T cells from patients with primary biliary cirrhosis, even when these latter cells exhibited sensitization to this same antigen complex. Using a component of the whole complex, the asialoglycoprotein receptor as antigen, migration inhibitory factor was invariably released by T cells from patients with autoimmune chronic active hepatitis, but from only 2 of 8 patients with primary biliary cirrhosis sensitized to the whole complex. Thus, in autoimmune chronic active hepatitis, but not in primary biliary cirrhosis, the asialoglycoprotein receptor is invariably a target for cellular immune reactions and is associated with a suppressor T-cell defect for hepatocyte antigens.

Human T cell subsets differ in their ability to migrate in vitro and to produce T cell migration inhibitory factor.

The spontaneous migration in vitro, production of T cell migration inhibitory factor (TIF) and the response to TIF of OKT4+ and OKT8+ human T cell subsets were studied. The OKT4+ lymphocytes migrated far better than the OKT8+ cells although the movement of both subsets was comparably inhibited by TIF. The OKT8+ subset was found to be a major source of TIF, while OKT4+ cells were responsible for macrophage migration inhibitory factor (MIF) production. The implications of lymphokine production by OKT8+ cells for the regulation of inflammatory responses are discussed.

Chemoattractant lymphokines specific for the helper/inducer T-lymphocyte subset.

The cellular content of T-lymphocyte-rich inflammatory sites is dependent in part on the in situ elaboration of chemoattractant factors. We have previously described three T-lymphocyte-specific chemoattractant lymphokines; a chemokinetic factor, lymphocyte chemoattractant factor (LCF, MW 56,000), and two distinct lymphocyte migration inhibitory factors (LyMIF75K, MW 75,000; and LyMIF35K, MW 35,000). These factors are produced by human T cells in response to antigen, concanavalin A, or histamine stimulation. In this communication, we report that LCF and LyMIF35K are produced by OKT8+ (suppressor/cytotoxic) and OKT4+ (helper/inducer) lymphocytes, respectively, and are selectively chemoattractant for the OKT4+ lymphocyte subset. LyMIF75K is produced by OKT4+ cells and inhibits both OKT4+ and OKT8+ lymphocyte migration. Production of LCF and LyMIF35K by infiltrating lymphocyte subsets may be one mechanism whereby unactivated helper/inducer T lymphocytes are selectively recruited to sites of inflammation.

[Studies of an assay system for dialyzable leukocyte extracts (DLE)--influence of DLE on leukocyte migration inhibition test by HBsAg].

In order to examine the suitability of leukocyte migration inhibition test (LMIT) in the capacity of in vitro assay system for dialyzable leukocyte extracts (DLE), the effect of DLE on hepatitis B and its antigen-specificity, the migration inhibitory activities to purified hepatitis B surface antigen (HBsAg) was measured using the leukocyte MIF test with DLEs obtained from HBsAb-positive or HBsAb-negative blood. The direct LMIT using agarose plate was modified according to the technique of Clausen et al. In spite of our assay system was dose-dependent for PPD, a significant response for purified HBsAg was not observed. However, some meaningful migration inhibition appeared when HBsAg and DLE were added simultaneously to the migration cells. From these results, it is concluded that DLE has antigen-specific and/or antigen non specific influences to the cell-mediated immunity for HBsAg Though some problems remain, we think our results are interesting, since the assay system for DLE has not been established and our study is closely related to the effect of DLE concerning hepatitis B.