Leukocyte Inhibitory Factor Research Articles

Deficient lymphokine production of newborn lymphocytes.

Lymphokine production by newborn lymphocytes was assessed by measuring migration inhibition factor (MIF) and leukocyte inhibition factor (LIF) of isolated mononuclear cells from cord blood, 1-7-days-old newborns, and adult controls. Ficoll-Hypaque separated mononuclear cells were stimulated with phytohemagglutinin (PHA) or allogeneic lymphocytes in a mixed leukocyte culture (MLC), and the supernatants were harvested at optimal times for lymphokine assays. Thymidine incorporation into DNA was also assayed to calculate a proliferative index. MIF was assessed by the inhibition of adult mononuclear phagocyte cell migration under agarose; LIF was assessed by polymorphonuclear cell migration under agarose. Although the proliferative responses of cord and newborn cells are equivalent or greater than those of adult controls, the PHA-induced MIF production in cord blood and newborn lymphocytes was only 46% and 12.5% respectively of mean adult levels; MLC-induced MIF production was 44% and 7%, respectively of mean adult levels. PHA-induced LIF production in cord blood was 27% of adult levels. These differences are only appreciated if dilutions of the supernatants are assayed. Simultaneous assay of MIF and LIF production in dilution of supernatants from adult lymphocytes showed higher LIF activity, whereas in cord lymphocytes MIF activity was greater than LIF activity. This further emphasizes the non-identity of MIF and LIF. These results indicate another abnormality of T cellular immunity in newborns not detected by T-cell enumeration or proliferative responses and parallels other defects in specialized T cell function such as cytotoxicity and immune interferon production.

Effects of leukocyte inhibitory factor (LIF) on human neutrophil function.

The effects of the lymphokine, leukocyte inhibitory factor (LIF), on human neutrophil function were studied. This soluble mediator, which is defined by its specific inhibition of neutrophil locomotion, does not interfere with chemotactic factor binding and does not affect basal or stimulated superoxide generation by neutrophils. In contrast, phagocytosis of opsonized Staphylococcus aureus is markedly inhibited by LIF, and degranulation is stimulated by this lymphokine. The possible mechanisms of LIF action on neutrophils are discussed.

Leukocyte inhibitory factor (LIF) in granuloma annulare: a comparative study between the generalized and the localized types

Leukocyte inhibitory factor (LIF) was investigated in 9 patients with localized granuloma annulare and 8 patients with the generalized form of the disease. The control group consisted of 10 matched, apparently healthy subjects. LIF values were significantly higher in patients with granuloma annulare of both groups than in controls (p less than 0.05 and 0.01). However, no significant difference was revealed between the two groups of patients. Though the results seem to speak for a cell-mediated immune response in granuloma annulare, they do not add a further support to the previously demonstrated differences between the two forms of granuloma annulare.

Behçet's disease leucocyte inhibitory factor (LIF) in response to skin extract.

ABSTRACTThis is a study of leucocyte inhibitory factor (LIF) in patients with Behçet's disease using an extract from a reactive (pathergic) lesion as an antigen and, as a control, an extract from normal skin of a normal subject. The purpose is to search for the possible existence of an antigen in the skin that may explain the development of this reactive lesion following a trivial trauma.The LIF was tested in 9 patients with the disease; in 7 both during remission and exacerbation of symptoms, and in 9 healthy matched controls. The results failed to indicate any existence of an antigen in the skin of the tested patients.

Physiochemical characterization of human histamine-induced suppressor factor

A product of histamine-stimulated human lymphocytes, histamine-induced suppressor factor or HSF, was characterized by enzyme treatment, sensitivity to reduction and alkylation, by molecular sieve chromatography, and by polyacrylamide gel electrophoresis. HSF was found to have a wide pH stability (pH 3–10), sensitivity to temperatures greater than 80 °C, and to have the properties of a glycoprotein by virtue of its sensitivity to chymotrypsin, trypsin, sodium periodate, and neuraminidase. HSF did not appear to have a serine group(s) in its “active” site since its biologic activity remained intact following treatment with an irreversible serine esterase inhibitor (phenylmethylsulfonyl fluoride). Further, HSF did not appear to have inter- or intramolecular disulfide linkages because treatment with denaturing and/or reducing agents, followed by alkylation, did not significantly alter its activity. Molecular sieve chromatography employing Sephadex G-100 revealed an apparent molecular weight for HSF of 25–40,000. Electrophoresis of HSF in polyacrylamide gels at pH 8.7 under nonreducing conditions revealed two regions of activity, one region migrating with albumin and the other region anodal to albumin. In addition to suppressing lymphocyte proliferation, the 25–40,000 M r, Sephadex G-100 fractions also inhibited the production of leukocyte inhibitory factor. Of particular interest, gel filtration of supernatants generated by stimulating mononuclear cells with either histamine, dimaprit (but not 2-pyridylethylamine), concanavalin A, or Candida albicans resulted in similar elution profiles with regard to inhibition of lymphocyte proliferation. That is, 25–40,000 M r fractions of supernatants generated by each stimulant suppressed lymphocyte proliferation to a similar degree. The latter findings provide indirect evidence that T lymphocytes, triggered in response to antigenspecific and nonspecific stimuli, elaborate suppressor molecules capable of modulating T-cell function that share certain similarities.

Lymphokine production and lymphocyte transformation by human mononuclear cells in a serum-free medium

In order to study the effects of purified serum proteins, such as complement components, on human lymphoid cell functions, we have developed a serum-free culture medium that supports lymphocyte transformation and lymphokine production. The medium is comprised of RPMI 1640 containing commercially available purified human serum albumin (HSA). Optimal proliferative responses to mitogens and antigens were noted at HSA concentrations of 2.5-10.0 mg/ml, and at a cell density of 1 X 10(6) mononuclear cells/ml. In medium supplemented with 5 mg/ml HSA, it was found that lymphocyte transformation responses, release of interleukin 2 and leukocyte inhibition factor (LIF), and the one-way mixed lymphocyte reaction proceeded in a manner analogous to that in plasma-supplemented cultures. Stimulation of LIF release by C3b was seen in HSA-supplemented cultures but not in serum-free conditions or in cultures containing 12% autologous plasma. This system can thus be used to examine cell-mediated immune responses without the interference of plasma proteins other than HSA.

Collagens Act as Ligands to Stimulate Human Monocytes to Produce Mononuclear Cell Factor (MCF) and Prostaglandins (PGE2)

Blood mononuclear cells from patients with rheumatoid arthritis produce the lymphokine, leukocyte inhibitory factor (LIF) in response to collagens in vitro, and blood monocytes release prostaglandins (PGE2) and a factor, mononuclear cell factor (MCF) which stimulates collagenase and PGE2 production by cultured synovial cells. We therefore examined the effect of collagens on the production of PGE2 and MCF. Blood mononuclear cells from 6 patients with rheumatoid arthritis and 6 normal subjects were cultured in native human types I, II, or III collagen-coated tubes, or with streptokinase-streptodornase (SK-SD), and the supernatant media derived from these cultures analyzed for the presence of MCF, PGE2, and LIF. Types II and III collagens, as well as SK-SD, markedly stimulated MCF production by the cells from all 12 subjects (MCF activity, expressed as a mean stimulation index (SI) ± SEM, was 43 ± 12 for type II, 33 ± 7 for type III, and 37 ± 23 for SK-SD). Type I collagen was less stimulatory (mean SI 10 ± 7). Cells from the patients with rheumatoid arthritis, but not the normal subjects, produced LIF in response to types II or III collagens but not to type I collagen. PGE2 production by blood mononuclear cells paralleled that of MCF, although abrogation of PGE2 release with indomethacin did not reduce MCF production. a chains purified from denatured collagens also stimulated MCF production. Using cells from patients with rheumatoid arthritis, type II collagen stimulated production of all three factors in the presence of polymyxin B or fibronectin-depleted serum, suggesting, respectively, that neither endotoxin nor fibronectin were responsible for their generation. Monocytes, purified from normal blood by an adherence tech

Modulation of human lymphocyte function by C3a and C3a(70-77).

Human C3a and the synthetic octapeptide C3a (70-77), which retains the activities of an anaphylatoxin, inhibit in a concentration-dependent manner the generation of leukocyte inhibitory factor (LIF) activity by human mononuclear leukocytes and T lymphocytes cultured with the mitogens phytohemagglutinin (PHA) or concanavalin A (Con A) or the antigen streptokinase-streptodornase (SK-SD). The generation of LIF activity was inhibited by 50% by 10(-8) M C3a or C3a(70-77) with PHA or Con A as the stimulus, whereas a more than 10-fold higher concentration of C3a(70-77) than C3a was required to achieve the same level of suppression with SK-SD as the stimulus. Similar concentrations of C3a(70-77) inhibited to the same extent the migration of T lymphocytes stimulated by alpha-thioglycerol of Con A. Neither C3a nor C3a(70-77) altered significantly the uptake of [3H]thymidine by human mononuclear cells exposed to PHA, Con A, or SK-SD. The capacity of C3a(70-77)-Sepharose,m but not Sepharose alone, to adsorb or inactivate mononuclear leukocytes required for the generation of LIF activity established a direct interaction. Analysis of the lymphocytes in the effluent from C3a(70-77)-Sepharose columns, using monoclonal antibodies to surface antigens, showed a selective depletion of the helper/inducer population of lymphocytes. C3a might represent an important mediator of the functionally selective regulation of human T lymphocyte activities by the complement system.

Lymphoid cell lines as indicators of lymphokine generation.

We investigated the migration characteristics of the cells of four human lymphoid lines, normal peripheral blood leucocytes (PBL), and normal mouse splenocytes (MSC). Two lines (QIMR-WIL and Namalwa) were actively migratory, as were the PBL and MSC. Migration was inhibited at low temperatures and reduced by inhibition of glycolysis, oxidative phosphorylation and RNA synthesis. The migration of QIMR-WIL cells, PBL and MSC but not Namalwa was inhibited by lymphokine-containing supernatants from phytohaemagglutinin-pulsed MSC or MSC stimulated by allogenic cells. The inhibitor of migration in the supernatants had properties similar to those of human leucocyte inhibition factor but distinct from those of macrophage inhibition factor. QIMR-WIL and normal human PBL were compared as indicators in an indirect leucocyte migration assay. QIMR-WIL cells were the more sensitive responder cells and are an abundant, stable, and uniform cell population for lymphokine detection.

Partial Characterization of Lymphokines that Change the Surface Charge Density of Human Polymorphonuclear Leukocytes

We have investigated the effects of stimulated human lymphocyte supernatants on the surface charge density of human polymorphonuclear leukocytes using analytical free-flow electrophoresis. Unfractionated ConA-induced human lymphocyte supernatants were found to decrease the electrophoretic mobility of PMN leukocytes by 10–16 %. Optimal conditions for the production of this lymphokine as well as for the PMN leukocyte-lymphokine interaction are described. In order to obtain more information about the factor responsible for the effect, active and control supernatants were fractionated by Sephacryl S-200 column chromatography, Sephadex G-50, and Amicon membrane filtration. The fractions obtained were dialysed, concentrated, and tested for activity on PMN leukocytes in free-flow electrophoresis. Fractions obtained in parallel were tested for Leukocyte Inhibition Factor (LIF) activity using the Clausen assay. Decrease in electrophoretic mobility of human PMN leukocytes was found to be due to two distinct lymphokines with molecular weights of about 10,000–20,000 and 50,000–60,000 d, respectively. These fractions showed no activity in the Clausen assay. Fraction V (60,000–80,000 d) revealed strong activity in the Clausen assay, but had no effect on the surface charge density of human PMN leukocytes. The ConA-induced lymphokines that changed the surface charge density of PMN leukocytes retained their activity after heating to 56°C for 30 min. They were not synthesized in the presence of puromycin, but mitomycin C treatment of the lymphocytes had no effect on the production of the lymphokines. Moreover, pretreatment of human PMN leukocytes with puromycin for 30 min blocked the reaction, indicating an active role of the PMN leukocytes following the reaction with the lymphokines. Finally these lymphokines preferentially influenced the surface charge density of human PMN leukocytes but not that of rat or guinea-pig peritoneal exudate cells.

Pyoderma Gangrenosum

Aberrations of cellular immune functions in pyoderma gangrenosum (PG) may lead to nonspecific activation of inflammatory cells or to an imbalance of suppression leading to autoaggression (chronic ulceration). A patient with severe unremitting PG had anergy to a battery of seven skin test antigens. Mixed lymphocyte reactions, autologous mixed lymphocyte reactions, lymphocyte proliferative responses to antigens, and the production of leukocyte inhibitory factor were substantially suppressed, while the lymphocyte responses to mitogens were unaffected. Quantitative immunoglobulin and complement levels were normal. The inhibition of cellular immune functions was mediated by a factor in the patient's serum. This factor also inhibited lymphocyte functions of normal unrelated control subjects. Preliminary studies demonstrated that the factor is nondialyzable, heat stable, and not adsorbed by Staphylococcus A protein. Pulse therapy with large doses of corticosteroids resulted in dramatic clinical improvement.

Isocyanate-induced pulmonary diseases: a current perspective

Chemicals of the isocyanate family are required for the production of a variety of commercial products, including insulation materials, automobile upholstery, furniture, and surface coatings. They are highly reactive with other chemicals containing hydrogen atoms. Early reports of medical toxicity appeared 25 yr ago and have been confirmed on a worldwide basis. Adverse respiratory symptoms have been the chief concern, and a wide spectrum of work-related lung diseases has been described. Although some of these reactions were undoubtedly due to primary irritation, the majority presented with airways obstructive abnormalities clearly identified as classic asthma. The possibility of an underlying hypersensitivity mechanism was suggested by the insidious onset of symptoms, a latency period of weeks to months, peripheral and tissue eosinophilia, and the elicitation of symptoms after exposure to very small subtoxic levels of these chemicals. Some cases consistent with the unique clinical features of hypersensitivity pneumonitis have also been recognized. Early animal experimental studies had suggested that immunologic factors were important etiologic components of these syndromes. However, when several independent groups of investigators could not confirm these preliminary results, the pathogenesis of these diseases soon became a highly controversial subject. Current experimental and clinical approaches have therefore been redirected to encompass the role of nonimmunologic mechanisms in the pathogenesis of adverse isocyanate reactions. Collectively, these recent investigations suggest that both immunologic and nonimmunologic mechanisms appear to be operative as causes of isocyanate-induced asthma. There is in vitro evidence both for and against possible specific pharmacologic effects of diisocyanate compounds. Parallel bronchoprovocation studies comparing the activities of diisocyanates, methacholine, and histamine suggest that diisocyanates may act either as direct pharmacologic agonists or as inducers of nonspecific bronchial reactivity. Recent evidence of immunoreactivity to both monofunctional and bifunctional isocyanates in several animal models and sensitized workers has also mandated a reassessment of immunologic factors. Hapten-specific, IgE-mediated antibody responses have been observed in both animal and human investigations. However, only a small subset of sensitized workers developed specific IgE antibodies to p-tolyl monoisocyanate and/or hexyl monoisocyanate. Both IgE and precipitating antibodies have been reported in several cases of workers sensitized to methylene diphenyl diisocyanate. Some toluene diisocyanate-sensitive patients also exhibit other types of immunologic responses, including antigen-induced lymphocyte transformation and synthesis of a leukocyte inhibitory factor. Detailed immunologic investigations have not yet been carried out in most of the suspected cases of hypersensitivity pneumonitis. Other possible nonimmunologic sequelae of isocyanate reactions include chronic bronchitis, recurrent pneumonia, pulmonary edema, and pulmonary emphysema. However, the question of chronic pulmonary disability associated with these diseases is poorly understood and requires further long-term investigation. Despite the brisk controversy that still exists about the pathogenesis of isocyanate-induced pulmonary diseases, new data derived from animal models and clinical investigation of sensitized workers suggest that application of several innovative approaches could result in early detection and treatment of clinical reactions. These are considered in the context of pre-employment screening, serial monitoring in the workplace, and postemployment surveillance.

Purification of leukocyte inhibitory factor derived from a human non-T, Non-B cell line

Purification of leukocyte inhibitory factor derived from a human non-T, Non-B cell line

Specific binding of leukocyte inhibitory factor to neutrophil plasma membranes.

The interaction of leukocyte inhibitory factor (LIF), a lymphokine that acts as a cell-directed inhibitor of neutrophil locomotion, with the plasma membrane of its target cell was studied. LIF bound specifically to intact neutrophils and to neutrophil plasma membrane vesicles but not to intact macrophages, erythrocytes, or plasma membrane vesicles obtained from these cells. Although LIF adhered tightly to the neutrophil plasma membrane and was not removed by extensive washing, LIF activity could be eluted with N-acetyl-D-glucosamine. Elution with galactose, fucose, and N-acetyl-D-galactosamine did not remove LIF from the neutrophil plasma membrane. Similarly, N-acetyl-D-glucosamine, but not galactose, interfered with the binding of LIF to neutrophil plasma membrane vesicles. It is proposed that LIF binds to a high-affinity neutrophil plasma membrane site and that some intrinsic neutrophil locomotory abnormalities may represent tightly bound inhibitors of migration.

Immunology of human gastric cancer: a preliminary report.

The immunological spectrum in fifteen patients with gastric cancer is presented. Patients were divided in three groups. Those with nonadvanced cancer, those with advanced but resectable lesions and those with advanced but nonresectable tumors. Preoperatively, elevated levels of circulating immune complexes (CIC) associated with hyporesponsiveness to phytohemagglutinin (PHA) and in mixed lymphocyte culture (MLC) as well as a positive leukocyte inhibitory serum factor (LIF-S) were found in nearly half of the patients. Inhibitory or enhancing autologous serum factors were detected. Postoperatively, immunologic parameters return to normal in patients with nonadvanced cancer, while in advanced cancer, antibody and cell-mediated immune response remained altered, with some changes associated with chemotherapy. These findings are probably related with the presence or absence of tumor and offer a distinct approach in evaluating the immunologic response of a tumor-bearing patient.

Serum factors inhibiting some leukocytic functions in Hodgkin's disease

The presence of leukocyte inhibitory factor(s) (S-LIF) in the serum of patients with Hodgkin's disease (HD) has been investigated. It was possible to demonstrate the inhibitory activity in 50% of the patients by employing the agarose plate method. The PHA response in these patients has been found to be reduced. A linear correlation exists between this reduction and the presence of S-LIF in the sera. In order to assess some biological properties of these sera, chemotaxis and phagocytosis of normal cells in the presence of sera from patients with HD have been investigated and both these leukocyte functions have been found to be reduced. The inhibitory capacity was detectable in the same sera which exhibited S-LIF activity and a close linear correlation could be shown. In order to assess whether S-LIF had the same properties of in vitro produced LIF, two monosaccharides were added: l-fucose, capable of blocking the inhibitory activity of LIF, and glucose, which does not block this activity. Surprisingly the addition of l-fucose and glucose greatly enhanced the inhibitory activity of S-LIF. These observations clarify the relationship between the presence of S-LIF and the immunological status in patients with HD and give some indications of the characterization of this (these) factor(s).

Purification of a lymphoid cell line product with leukocyte inhibitory factor activity.

Leukocyte inhibitory factor (LIF), a lymphokine that inhibits the random and directed migration of polymorphonuclear (PMN) leukocytes, was purified from a human non-T, non-B leukemia cell line (Reh). From 10 liters of serum-free supernatant, 1.3 microgram of protein with LIF activity was obtained by the sequential use of affinity chromatography with concanavalin A-Sepharose, hydrophobic chromatography with hexylagarose, and gel filtration chromatography. The specific activity of LIF recovered represented an 80,000-fold purification over that of the initial crude serum-free supernatants, and the preparation at that point was estimated to be 80--90% pure. To both assess the purity of the preparation and provide a further purification step, Reh LIF activity recovered by the above procedures was subjected to isoelectric focusing. One major stainable protein band was identified; its isoelectric point was pH 5.4--5.5. Gels run in parallel for recovery of biologic activity revealed only one region (pH 5.4--5.5) with ability to inhibit PMN leukocyte migration. Iodination of Reh LIF resulted in a loss of biologic activity, but isoelectric focusing of this material revealed one major 125I-labeled band (pH 5.1) and several minor bands. The coincidence of biologic LIF activity with one stainable protein band as identified by isoelectric focusing implies that the final product may be homogeneous.

Rat leukocyte inhibitory factor (LIF): Similarities to human LIF and generation by cells from rats with collagen-induced arthritis

Rat lymph node cells (LNC) produce a mediator which exhibits functional and physicochemical similarities to human leukocyte inhibitory factor (LIF). Measurement of LIF can be used to quantify cellular sensitivity in rats to foreign protein antigens or to a heterologous antigen in vitro. Concanavalin A-induced rat LIF has a molecular weight of 100,000-60,000 daltons and retains activity after heating to 56 °C for 30 min. Rat LIF is not synthesized in the presence of puromycin and appears to be a protein, since it is inactivated by treatment with chymotrypsin. Moreover, the activity of rat LIF is susceptible to the serine esterase inhibitor, diisopropylphosphofluoridate, but it is resistant to neuraminidase treatment. Finally, rat LIF preferentially inhibits the migration of rat and human polymorphonuclear leukocytes but not that of rat or guinea pig peritoneal exudate cells enriched for macrophages. Except for the more dispersed size of rat LIF, these properties are analogous to those described for human LIF. LIF activity is generated, in an antigen-specific manner, by LNC sensitized to ovalbumin or purified protein derivative of tuberculin when cultured with the antigen used for sensitization. LNC from rats rendered arthritic by prior intradermal (id) injection of native chick type II collagen in incomplete Freund's adjuvant produce LIF in response to this heterologous antigen. These studies delineate a new assay for cellular sensitivity in rats and provide additional evidence that cellular reactivity to type II collagen is present in this animal model of arthritis.

Diverse profiles of immunoreactivity in toluene diisocyanate (TDI) asthma.

Possible immunoreactivity to chemically well-characterized mono- and diisocyanate protein conjugates was reevaluated in 15 workers with TDI asthma and 17 normal (nonexposed) volunteers. Lymphocytes of nine sensitive workers were incubated with TDI human serum albumin (HSA) conjugates. Leucocyte inhibitory factor (LIF) was produced. Leucocyte inhibitory factor was also induced by hexamethylene diisocyanate (HDI) protein conjugates in four of these workers who had no prior history of exposure to HDI. Disappearance of TDI- and HDI-induced LIF was noted in several sensitive workers who were removed from further TDI exposure. Three LIF-positive workers also demonstrated positive intracutaneous reactivity to TDI-HSA. One workers had a markedly positive RAST (25.5% binding) to a monofunctional (p-tolyl isocyanate) protein reagent. These studies suggest that isocyanates have the potential for eliciting heterogeneous immune responses in certain subpopulations of exposed workers. Continued contact with isocyanates may be necessary for maintenance of specific immunity. Possible cross reactivity between TDI and HDI may be determined by new antigenic sites created by isocyanate protein interactions.

Partial characterization of a lymphoid cell line (Reh) product with leukocyte inhibitory factor (LIF) activity.

Partial characterization of a lymphoid cell line (Reh) product with leukocyte inhibitory factor (LIF) activity.