Leukocyte Migration Inhibitory Factor Activity Research Articles

Pathophysiological significance of a reaction in mouse gastrointestinal tract associated with delayed-type hypersensitivity.

To explore the pathophysiological significance of delayed type hypersensitivity (DTH) reaction in mouse gastrointestinal tract induced by an allergen 2,4-dinitrochlorobenzene (DNCB). BALB/c mice were randomly divided into control and DTH(1-6) groups. After sensitized by DNCB smeared on the abdominal skin, the mice were challenged with DNCB by gavage or enema. The weight, stool viscosity and hematochezia were observed and accumulated as disease active index (DAI) score; the gastrointestinal motility was represented by active charcoal propulsion rate; the colon pathological score was achieved by macropathology and HE staining of section prepared for microscopy; and the leukocyte migration inhibitory factor (LMIF) activity was determined by indirect capillary assay of the absorbance (A) of migrated leukocytes. Active charcoal propulsion rates of small intestine in the DNCB gavages groups were significantly higher than that in the control group (P<0.01). The DAI scores and pathological score in DNCB enema groups were also higher than that in the control group (P<0.05), and there were significant rises in LMIF activity in DNCB enema groups as compared with control groups (P<0.01). Mouse gastrointestinal DTH reaction could be induced by DNCB, which might facilitate the mechanism underlying the ulcerative colitis.

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.

Leukocyte migration inhibition studies and neutrophil cell function during aging in Nigerians

In this study, in vitro cell-mediated immune response was analysed in 150 healthy Nigerians between 6 and 95 years old by the leukocyte migration inhibitory factor (L-MIF) test. Lymphocytes were activated with the mitogen concanavalin A and candida, measles virus and mycobacterial antigens. Nonspecific cellular immune capacity was studied by the Nitroblue tetrazolium (NBT) test. Numerical estimates of leukocytes and lymphocyte subpopulations were done. Mean L-MIF activity obtained with the four lymphocyte activators decreased with rising age indicating a progressive decline in cellular immune function with age. There was no significant age-related change in formazan positivity rate for both unstimulated and stimulated NBT tests. No age-related change in number was observed for any of the leucocyte and lymphocyte subpopulations. These results show that cell-mediated response in Nigerians immunity declines, but phagocyte function is unchanged during aging. Lymphocyte depletion or numerical alteration in resting T cell subsets could not be demonstrated to be responsible for depressed cell-mediated immunity in aging Nigerians.

Synthetic peptides comprising defined sequences of CH-2 and CH-3 domains of human IgG1 induce prostaglandin E2 production from human peripheral blood mononuclear cells.

Synthetic peptides Y48 and Y75 comprising sequences at exposed sites within the CH-2 and CH-3 domains of human IgG1 at a concentration of 10(-5) M, increase PGE2 production by human peripheral blood mononuclear cell (PBMC) cultures. An increase of leukocyte migration inhibitory factor (LMIF) production in PBMC cultures--as a result of synthetic peptide treatment--was also observed. This LMIF activity, to some extent, is attributed to the PGE2 production by the cells; the inhibition of leukocyte migration being abolished by the presence of indomethacine or antibody to PGE2.

Demonstration of leukocyte inhibitory factor in human schistosomiasis mansoni and its evaluation from patients in an endemic setting

Leukocyte migration inhibitory Factor (LIF) is produced by the peripheral blood mononuclear cells (PBMN) of most patients with Schistosoma mansoni infection upon their exposure to soluble egg antigens (SEA). PBMN of some patients also respond to adult worm (SWAP) antigens by LIF production. LIF is stable at 37°C for 60 min but is sensitive to heating at 56°C for even 30 min. The serine protease inhibitor PMSF destroyed LIF activity at concentrations of 10 −2 to 10 −3 M. Concanvalin A stimulated production of detectable levels of LIF by 8 hr, while SEA and SWAP did so by 15 and 39 hr, respectively. PBMN of healthy normal controls did not produce LIF upon exposure to SEA or SWAP. PBMN of a few field controls (stool negative subjects from an endemic area) produced detectable LIF activity when exposed to SEA or SWAP. PBMN from most infected (stool positive) patients from an endemic area produced LIF when exposed to SEA and only occasionally did so to SWAP. Previous studies have shown that most often only the PBMN of former, cured patients, and not chronically infected patients, produce the lymphokine activity termed mitogenic factor (MF). The current data indicate that because LIF is primarily produced by actively infected patients, its production may be controlled by different immunoregulatory mechanisms. Furthermore, although most SEA-related responses are highly immunoregulated in active, chronically infected patients, SEA appears to be a better stimulus for patient PBMN production of LIF than SWAP.

Influence of in vitro irradiation upon lif production by cona stimulated mononuclear cells

Leukocyte migration inhibitory factor (LIF) activity of culture supernatants of in vitro irradiated Concanavalin A (ConA) stimulated lymphocytes was tested by measuring granulocyte migration from clotted plasma droplets placed in flat bottom microplates. The specificity of inhibition was assured by pretreating the assay supernatants with anti-LIF antibodies which abrogated granulocyte migration inhibition but did not impair guinea pig Peritoneal Exudate Cells (PEC) migration inhibition. In vitro irradiation (150–1200 rad) of MNC cultures either before or after ConA stimulation did not impair lymphokine production and sometimes significantly improved the supernatants' LIF activity as compared with that of unirradiated cultures. The existence of radiosensitive suppressor cells regulating LIF production by ConA stimulated mononuclear cells is suggested.

Activation of Fc receptor-bearing lymphocytes by immune complexes. II. Killer lymphocytes mediate Fc ligand-induced lymphokine production.

Cells that participate in immune complex-induced production of leukocyte migration inhibitory factor (LIF) activity can be concentrated in a population making up 2-4% of human peripheral blood lymphocytes in which greater than 90% of the cells are active in a single cell antibody-dependent cellular cytotoxicity assay. When so concentrated, such killer (K) cell preparations are as efficient in producing LIF activity as mitogen activated T lymphocytes. Other Fc receptor (FcR)-bearing lymphocytes, including natural killer (NK) cells, do not produce measurable LIF activity when incubated with immune complexes (additional evidence that the K and NK cells among ligands to the FcR of the appropriate lymphocytes, possibly without need for exogenous receptor bridging, is the only requirement for their activation to immune complex-induced lymphokine production (ICLP). It is probable that ICLP by K cells palays a role in antibody-mediated effector functions in vivo.

Improvement of LIF release by mononuclear cell cultures by 24 h incubation before stimulation with ConA

By measuring granulocyte migration from clotted plasma droplets placed in Microtest II plates, leucocyte migration inhibitory factor (LIF) production by concanavalin A (ConA) stimulated mononuclear cell (MNC) cultures was tested. Moderate LIF production was observed when mononuclear cells were pulsed with ConA at O h, as compared with cells stimulated after 24 h culture which produced a significantly increased amount of LIF. Fresh adherent cells, when added to 24 h incubated MNC cultures before ConA stimulation, decreased LIF production, in contrast to in vitro irradiated or overnight cultured adherent cells. Indomethacin, when present during exposure to a ConA pulse of either MNC or of adherent cell-rich 24 h incubated MNC cultures, improved LIF activity significantly. This suggests that prostaglandin producing adherent cells, presumably cooperating with radiosensitive short-lived immunoregulatory T cells, are involved in suppression of LIF production by MNC cultures stimulated by ConA at O h.

Spontaneous release of leucocyte migration-inhibitory factor by mononuclear cells eluted from rheumatoid synovial tissue.

Spontaneous leucocyte migration-inhibitory factor (LIF) was assayed by an indirect system in which mononuclear cells (MNC) eluted from rheumatoid synovial tissues or isolated from peripheral blood of normal donors were cultured without antigen. The supernatants from these cultures and control supernatants, heated to 80 degrees C for 30 min to destroy the LIF activity, were used in the test. In seven out of eleven rheumatoid arthritis patients the eluted synovial MNC produced LIF spontaneously, whereas none of the twelve normal blood donors showed any such production. The ability to show spontaneous LIF production was primarily seen in the joint tissues from sero-negative patients.

Use of benzoyl-L-phenylalanyl-L-valyl-L-arginine (3H) methyl ester as a sensitive and selective substrate for the human lymphokine, leukocyte migration inhibitory factor (LIF).

Human leukocyte migration inhibitory factor (LIF) appears to be a serine esterase and protease exhibiting specific affinity towards arginine esters and amides. On the basis of indirect evidence that an amide of the oligopeptide benzoyl-phenylalanyl-valyl-arginine might have high and selective affinity for LIF, we prepared an ester of this oligopeptide, benzoyl-phenylalanyl-valyl-arginine (3H) methyl ester (3H-BPVAME) for the direct measurement of LIF activity in a double-phase radio-enzyme assay. The hydrolysis of 3H-BPVAME followed enzyme-substrate kinetics in that the reaction was time-, temperature-, pH-, and concentration-dependent. 3H-BPVAME rpoved to be more selective and approximately 20 times more sensitive as a substrate for LIF than previously used radiolabeled substrates such as 3H-BAEE and 3H-TAME. Moreover, hydrolysis of 3H-BPVAME by partially purified LIF preparations was significantly inhibited by 10(-8) to 10 (-5) M of guanosine 3',5'-cyclic monophosphate (cGMP), further supporting the hypothesis that cGMP acts as a regulator of LIF activity. Inhibition of LIF-induced esterolysis was also provided by dibutyryl cGMP, but only at concentrations 10(-7) to 10(-5) M; 8-bromo cGMP and adenosine 3',5'-cyclic monophosphate were both ineffective. These results provide support for the use of 3H-BPVAME as a more selective substrate to detect esterase activity in LIF preparations than heretofore described and the possible development of a biochemical assay for the measurement of this lymphokine.

An indirect assay for canine leukocyte migration inhibitory factor (LIF): Demonstration of its species specificity

An indirect assay in agarose-gel was developed to measure secretion of leukocyte migration inhibitory factor (LIF) by canine lymphocytes. Supernatants from cultures of mitogen- or alloantigen-stimulated canine peripheral blood lymphocytes were assayed for LIF activity using purified granulocytes as indicator cells. Canine LIF activity could be detected provided either autologous or allogeneic canine granulocytes were used as indicator cells. In contrast, canine LIF uniformly failed to inhibit the migration of human granulocytes. Likewise, human LIF inhibited the migration of human but not canine granulocytes in this assay. The data indicated that this species specificity could be due to differences in the structures of LIF receptors on canine and human granulocytes.

Prostaglandin effect on lymphokine production in multiple sclerosis

Production of the lymphokine LIF (leukocyte migration inhibitory factor) by normal lymphocytes on stimulation with mitogens in inhibited by E type prostaglandins (PGE). It has been reported that the leukocytes of patients with multiple sclerosis (MS) are resistant to this inhibitory effect. We have studied 10 MS patients using an indirect technique for measuring LIF activity. This allows separation of the effects of PGE on lymphocyte LIF production from the effects on the action of LIF on the target granulocytes. There was no difference between normal subjects and MS patients in the effects of prostaglandins E1 and E2 in concentrations of 2.5 and 0.25 microgram per milliliter, on LIF production, or on granulocyte response to LIF. These results do not support the hypothesis that the chronic inflammatory process in MS is the result of resistance to the normal inhibitory effects of PGE on the cellular immune response.

The indirect assay for leukocyte migration inhibitory factor (LIF) — Standardization and the effect of pH

In the indirect assay for leukocyte migration inhibitory factor (LIF), lymphokine-rich supernatants were obtained by culture of stimulated lymphocytes and then tested for LIF activity in agarose plates using purified granulocytes as target cells. Studies on the standardization of the conditions under which LIF acts on the target cells are described, with emphasis on the use of ‘standard’ supernatants of known LIF activity and the influence of pH on the action of LIF and the sensitivity of the assay. The observation that LIF activity is reduced when the ambient pH falls below 7.2 is suggested as an explanation firstly for the ‘escape’ phenomenon seen particularly in capillary tube assays for LIF, and secondly for the reduced sensitivity of the capillary tube assay in comparison with the corresponding agarose plate assay.

Cell-Mediated Immunity to Pityrosporum Orbiculare in Tinea Versicolor

Pityrosporum orbiculare, the presumed etiologic agent of tinea versicolor, was cultured in vitro and antigenic extracts prepared from the cultured organisms. Studies with lymphocytes from human cord blood and peripheral blood of guinea pigs demonstrated that such extracts were not mitogenic. Further studies in guinea pigs indicated that the animals could be sensitized by the injection of P. orbiculare extract in Freund's complete adjuvant and that this extract could elicit lymphocyte transformation and delayed skin test responses in sensitized animals. A group of 12 tinea versicolor patients and 15 normal subjects were studied in vitro for cell-mediated immunity to P. orbiculare extract. The majority of the subjects tested in both groups demonstrated positive lymphocyte transformation responses to this extract, as well as to standard mitogens and common microbial antigens. However, lymphocytes from tinea versicolor patients produced significantly less leukocyte migration inhibitory factor activity when stimulated by Candida albicans and P. orbiculare extracts than did lymphocytes from normal subjects. This was also true if only subjects with positive lymphocyte transformation responses to these antigens were considered. Leukocyte migration inhibitory factor responses to streptokinase/streptodornase were not significantly different between the two groups. Therefore, it appears that although both normal subjects and tinea versicolor patients demonstrate prior sensitization to antigens of P. orbiculare, the effector function of lymphocytes from most tinea versicolor patients appears to be impaired in that they produce subnormal amounts of the mediator leukocyte migration inhibitory factor when stimulated with antigenic extracts of this organism.

Human Leukocyte Migration Inhibitory Factor (LIF)

Recently reported experiments suggest that human leukocyte migration inhibitory factor (LIF) has properties of an esterase and a protease with substrate specificities directed against arginine esters and amides. Also reported previously, the synthetic phosphodiester bis-p-nitrophenyl phosphate (BNPP) but not various phosphomonoesters preserve LIF activity in the presence of the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF). In this paper I demonstrate that guanosine 3'5'-cyclic monophosphoric acid (3',5'-cGMP), a naturally occurring phosphodiester, at concentrations in excess of 10(-5)M also protects LIF against PMSF inactivation. The effect seems specific for the diester bond, its position in the nucleotide, and the guanine base. The possibility that LIF may be a multifunctional or an allosteric enzyme regulated by 3',5'-cGMP is discussed.

MODULATION OF HUMAN LEUCOCYTE MIGRATION INHIBITORY FACTOR (LIF) BY 3', 5'‐CYCLIC AMP, 3', 5'‐CYCLIC GMP AND AGENTS KNOWN TO INFLUENCE INTRACELLULAR CYCLIC NUCLEOTIDE METABOLISM

A study was made of the effects of 3', 5'‐cycylic AMP (3', 5' cAMP), 3', 5'‐cyclic GMP (3', 5' cGMP) and their dibutyryl derivatives, and the effects of various both cyclic and non‐cyclic nucleotides and of nucleosides on the in vitro migration of human peripheral blood leucocytes under agarose and on the activity of leucocyte migration inhibitory factor (LIF). Leucocyte migration was not significantly influenced by any of the above‐mentioned drugs. However, LIF activity was significantly depressed by 3', 5' cAMP and dibutyryl 3', 5' cAMP, whereas cells challenged with 10‐4M of the other drugs, including 2', 3' cAMP, 3'‐AMP, 5'‐AMP and adenosine, showed unreduced migration inhibition under standard test conditions. A possible role of 3', 5' cAMP in the mechanism of LIF action was supported further by experiments with various drugs known to influence intracellular 3', 5' cAMP metabolism. Treatment of leucocytes with the 3', 5' cAMP generating, beta‐adrenergic agent isoproterenol (10‐4M) caused a rapid, transient reduction of LIF activity as compared to LIF‐treated controls. The alfa‐adrenergic agent norepinephrine (10‐4M) was ineffective. Treatment of leucocytes with the phosphodiesterase inhibitors papaverine (10‐4M) and dipyridamole (2×10‐5) enhanced their motility and enabled them to escape migration inhibition as compared to LIF‐treated controls. 3', 5' cGMP may also participate in the expression of LIF activity, since cells treated with 3', 5' cGMP partially escaped migration inhibition during the first 3 hours of migration.

Human Leukocyte Migration Inhibitory Factor (LIF)

Previous findings suggesting an esterase and protease nature of human leukocyte migration inhibitory factor (LIF) were extended by testing the ability of different protease and esterase inhibitors to reduce LIF activity. The serine-specific inhibitors phenylmethylsulfonyl fluoride (PMSF) and physostigmine (eserine) markedly reduced LIF activity, whereas the histidine-specific inhibitors N-tosyl-L-lysine chloromethyl ketone (TLCK) and L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) were inactive. That LIF might act as an esterase and a protease was further strengthened by the ability of pralidoxime methansulfonate (2-PAM) to reestablish LIF activity of supernatants previously inactivated by PMSF. Furthermore, the arginine amides N-benzoyl-L-arginine-p-nitroanilide (BApNA) and N-benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (BPVApNA) were shown to satisfy the substrate specificities of the putative LIF enzyme. Indeed, BPVApNA seemed to possess a particularly strong affinity for LIF, indicating its potential role in an enzymatic LIF assay.

The production of heterologous antibodies to the human lymphokine: Leukocyte migration inhibitory factor (LIF)

Human leukocyte migration inhibitory factor (LIF) produced by concanavalin A-stimulated lymphocytes was partially purified by Sephadex G-100 chromatography and immunosorption of protein contaminants. This material was injected into two rabbits, and the IgG-IgA fractions of the resulting antisera (anti-LIF) neutralized LIF induced by antigen (PPD tuberculin) with as equal efficiency as that of LIF induced by mitogen. Anti-LIF activity was neither removed by absorption with control supernatant or normal human serum nor was it suppressed by absorption with lymphocytes or lymphoblasts. On the other hand, antibodies against human lymphoid cells (ALG) did not reduce LIF activity, indicating the difference between anti-LIF and classical ALG. In support of this, anti-LIF, in contrast to ALG, was not cytotoxic to lymphocytes, and it did not inhibit spontaneous T-rosette formation with sheep erythrocytes. In crossed immunoelectrophoresis using a conventional proteinstaining technique, only three precipitates appeared. None of these contained LIF. However, a protein migrating in the prealbumin region appeared to be specific for lymphocyte stimulation. The nature and significance of this product is unknown.

IN VITRO MODULATION OF HUMAN LEUCOCYTE MIGRATION AND MIGRATION INHIBITORY FACTOR (LIF) ACTIVITY BY CYCLIC 3‘, 5’‐AMP AND CYCLIC 3‘, 5’‐GMP

The effects of cyclic 3', 5'-AMP (cAMP) and cyclic 3', 5'-GMP (cGMP) on the in vitro migration of human peripheral blood leucocytes under agarose and on the activity of leucocyte migration inhibitory factor (LIF) was studied. Leucocyte migration was not influenced by dibutyryl cAMP, while the dibutyryl derivative of cGMP significantly stimulated cell migration (1 x 10(-5)M). LIF-treated leucocytes partially escaped migration inhibition in the presence of dibutyryl cAMP (greater than or equal to 1 x 10(-6)M), while dibutyryl cGMP was inefficient. If the parent compounds cAMP and cGMP were tested, almost similar results would be obtained, although at higher concentrations of the drugs. These results represent initial experiments with a view to investigating the possible role of cyclic nucleotides in the expression of LIF activity.

Human Leukocyte Migration Inhibitory Factor (LIF)

The activity of leukocyte migration inhibitory factor (LIF) obtained from Sephadex‐G‐100‐chromatographed supernatants of concanavalin‐A‐stimulated human lymphocytes was suppressed by two synthetic serine esterase and serine protease inhibitors (di‐isopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF)). LIF activity was also reduced by the naturally occurring protease inhibitors soybean trypsin inhibitor and aprotinin. The observed effect of DFP and PMSF was irreversible, since elimination of the inhibitors by dialysis did not restore LIF activity. The effect of PMSF was dose‐, time‐, and temperature‐dependent, and hydrolytic products of PMSF as well as sodium fluoride were inactive in blocking LIF. These results suggest that LIF may act as a serine esterase or a serine protease, or both of these, and that this putative enzyme is present in an activated form in supernatants from mitogen‐stimulated mononuclear cells.