Production Of Leukocyte Inhibitory Factor Research Articles

The G->c polymorphism of the p53 gene is strongly associated with decreased pregnancy rates in fresh IVF patients via an ovarian mechanism

OBJECTIVE: p53 is a key tumor suppressor gene responsible for the repair of damaged DNA. A polymorphism of the p53 gene at codon 72 (Arg→Pro) decreases p53 activity and may lead to an increase in DNA damage during oogenesis. Protein products of the p53 gene have also been shown to promote leukocyte inhibitory factor (LIF) production in the murine model and its reduction in p53 null mice significantly decreases litter size. We hypothesize that women who carry the p53c72 (pro/pro) polymorphism of the p53 gene may experience decreased implantation rates, either through diminished LIF production at the level of the endometrium or through diminished oocyte quality from inadequate DNA repair mechanisms. DESIGN: Prospective analysis of oocyte donor recipients and young women (<35) with unexplained infertility. Incidence of the p53c72 Pro (variant) polymorphism compared to p53c72 Arg (wild type) was assessed as well as IVF outcome. MATERIALS AND METHODS: A total of one hundred and sixty three samples were collected from donor egg recipients (DER) (n=76) and patients <35 years of age (n=87) with unexplained infertility. DNA was extracted and polymerase chain reaction amplification of p53c72 was performed. The frequency of each variant was compared to known population controls. Oocyte yield, implantation rates, and pregnancy rates were analyzed. RESULTS: The prevalence of the variant polymorphism was increased in our infertile population compared to the general population (47% vs. 35%, p<0.05). In the DER group, there was no difference in average age, implantation rates (52% vs. 48%) or pregnancy rates (78% vs. 85%) for patients with the variant polymorphism compared to the wild type, respectively. In the fresh IVF group, there was no difference baseline characteristics, however, within this group those with the polymorphism indicating lower p53 activity had decreased oocyte yield (9.31± 4.1 vs.12.8±5.3, p<0.05), decreased implantation rates (16% vs. 44%, p<0.01), and decreased pregnancy rates 30% vs.68% p<0.01) compared to the wild type. CONCLUSIONS: We have found that women who are homozygous for the p53c72Pro variant have both a decreased ovarian response to exogenous gonadotropins and decreased pregnancy rates when compared to the wild type. This suggests that p53 activity is crucial to oogenesis and early embryogenesis. The lack of impact of p53 activity on the DER population suggests that other cytokines may help overcome any negative impact of this polymorphism at the level of the endometrium.

Leukocyte Migration Inhibition in Amiodarone-Associated Pneumonitis

Amiodarone-associated pneumonitis is now a well-known clinical entity, but the mechanism for the induction of the pulmonary disease is ill defined. In four patients with this disorder, evidence was obtained for elaboration of a lymphokine, leukocyte inhibitory factor (LIF), by peripheral blood lymphocytes after incubation with amiodarone in the direct leukocyte migration inhibition test. Control lymphocytes from normal subjects, as well as from patients receiving amiodarone but without pneumonitis, failed to elaborate LIF in the presence of the drug in this test. This production of LIF suggests that pneumonitis associated with amiodarone therapy is also associated with a specific cellular immune response to the drug.

Leukocyte Migration Inhibition in Methotrexate-Induced Pneumonitis: Evidence for an Immunologic Cell-Mediated Mechanism

Methotrexate-induced pneumonitis is a well-known clinical entity, but the mechanism for the induction of the pulmonary disease is ill defined. In three patients with this disorder, evidence was obtained for elaboration of a lymphokine, leukocyte inhibitory factor (LIF), by peripheral blood lymphocytes after incubation with methotrexate (MTX) in the direct leukocyte migration inhibition test. Control lymphocytes from normal subjects, as well as from patients receiving methotrexate but without pneumonitis, failed to elaborate LIF in the presence of the drug in this test. Along with these results, we obtained bronchoalveolar lavage (BAL) cell data displaying high grade lymphocyte alveolitis with a lymphocyte subset inverted ratio. This production of LIF suggests that pneumonitis associated with methotrexate therapy is also associated with a specific cellular immune response to the drug.

Cellular Immunity to Sperm in Infertile Women

To investigate further whether immunity to sperm contributes to some cases of infertility in women, the production of leukocyte inhibitory factor by blood mononuclear cells challenged with human sperm in vitro was measured in women with infertility associated with poor results of postcoital tests or long-term unexplained infertility. Five (36%) of 14 infertile patients but none of nine fertile volunteers responded significantly to sperm in this assay. No study subject had serum IgG or IgM antibodies to sperm detected. These data provide additional evidence consistent with the possibility that cellular immunity to sperm-specific antigen(s) functions in the primary pathogenesis of infertility in some women.

1019 AUTOIMMUNITY TO ARTICULAR ANTIGENS IN JUVENILE ARTHRITIS

We studied 103 JA children, 46 controls and 25 patients with osteoarticular syndromes (OAS) to determine if differences in immune reactivity characterized different onsets or outcomes. We measured cell-mediated immunity (CMI) to human collagen, proteoglycan monomer (PM) and link protein (LP). Production of leukocyte inhibitory factor by mononuclear leukocytes was assayed and correlated with HLA type. Antibodies to native type II collagen were measured in an enzyme-linked immunosorbent assay. Results were analyzed using Fisher's Exact Test. JA patients were less reactive to ConA than controls, yet were more reactive to collagen I, II and PM. JA patients did not differ from OAS patients, CMI did not correlate with onset, course, out come, ANA, RF or HLA type. Collagen antibodies were found in 23/86 JA, 1/20 OAS, and 0/31 controls (p<0.01). These antibodies, confirmed by absorption studies, distinguished erosive disease from other JA (p<0.001). CMI to articular antigens may be of pathogenetic importance in JA, but in vitro reactivity does not predict risks or complications. The presence of antibodies to native type II collagen may be of greater importance in signaling poor outcome in JA.

Leukocyte migration inhibition in recurrent aphthous ulceration.

Leukocyte inhibitory factor (LIF) production in response to streptococcal antigens and oral mucosa was examined as a possible pathogenic mechanism in recurrent aphthous ulceration (RAU). Mononuclear cells from RAU patients and controls were stimulated with antigens prepared from two species of streptococci and from oral mucosa. Candida albicans and foreskin were tested as control antigens. There were some statistically significant differences in leukocyte migration inhibition between patients and controls with some concentrations of Streptococcus sanguis (ATCC 10556) and C. albicans, and a trend toward low migration indices with oral mucosa. However, the roughly equal numbers of patients and controls responding positively to each antigen and the large background variation suggest a lack of biological significance. LIF production did not increase consistently in the early stages and did not correlate with ulcer development in patients studied sequentially. We conclude that LIF production (at least in response to the antigens tested) plays no pathogenic role in RAU. Our data do not support the hypothesis that a specific cell-mediated sensitivity to streptococcal antigens is involved in RAU.

Lymphokine production and lymphocyte subpopulations in patients with head and neck squamous carcinoma.

Prior studies of impaired cellular immune reactivity in patients with head and neck squamous carcinoma (HNSC) suggest that immune deficiency associated with tumor growth may be related, in part, to alterations in immunoregulatory functions. To determine if production of soluble mediators of the immune response (lymphokines) is impaired in patients with HNSC, leukocyte migration inhibition in response to phytohemagglutinin was assessed in 32 patients with HNSC and 29 normal subjects and was correlated with levels of specific peripheral blood lymphocyte subpopulations to determine if quantitative levels of immunoregulatory lymphocyte subpopulations were related to in vitro lymphokine production. In the patients with cancer, leukocyte inhibitory factor production was consistently and significantly impaired and was directly related to levels of T4-positive (helper/inducer) lymphocytes. Substantial differences in levels of individual subpopulations were not detected among normal subjects and patients with cancer; however, the mean T4/T8 ratio was substantially increased in the patients with cancer. The findings confirm and extend prior observations of impaired cellular immune mechanisms in patients with HNSC and suggest that impaired lymphokine production may be related to quantitative and qualitative alterations in lymphocyte subpopulations.

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.

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.

Indirect Leukocyte Migration Inhibition Reactions to a 3-&lt;italic&gt;M&lt;/italic&gt; KCI Extract of Lung Adenocarcinoma by Lung Cancer Patients&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;

Mononuclear (MN) cells from the peripheral blood of lung cancer patients were tested for their ability to respond to a 3-M KCl extract of adenocarcinoma of lung with the use of an indirect leukocyte migration inhibition (LMl) assay. Antigen-stimulated MN cell cultures were evaluated for leukocyte inhibitory factor production by their ability to inhibit the migration of indicator polymorphonuclear cells from agarose droplets. When supernatants were prepared in conventional round-bottomed tubes (5X10(6) cell/tube), 25 of 44 (57%) lung cancer patients had positive indirect LMl responses to the 7661 antigen as compared to only 2 of the 30 (7%) normal donors. When supernatants were prepared in conical microtubes, with 10 times fewer MN cells, similar results were obtained. Patients with all histologic types of lung cancer had a similar incidence of reactivity, and reactivity of untreated patients did not appear to be related to stage of disease or degree of tumor burden. Surgical removal of the tumor appeared to decrease the incidence of reactivity in the 1- to 12-month postoperative period. These results strongly suggest that the LMl reactivity against lung tumor extracts is lymphokine mediated, inducing cellular responses by the patients against such antigens.

Comparative in vitro imunotoxicology of acyclovir and other antiviral agents

In vitro lymphocyte blastogenic responses to the commonly employed mitogens phytohemagglutinin, pokeweed, and concanavalin A were evaluated when acyclovir, adenine arabinoside, cytosine arabinoside, and idoxuridine were added to the culture materials. Similarly, specific antigen-induced blastogenic responses, including herpes group antigens, and cytotoxicity and leukocyte inhibitory factor assays with herpes group viruses were determined in the presence and absence of antiviral agents. No depression of these cellular immmune responses by acyclovir or adenine arabinoside ws demonstrated. This was in contrast to the effects of cytosine arabinoside and idoxuridine, which severely inhibited blastogenic and cytotoxic responses but not leukocyte inhibitory factor production. Even at concentrations up to 20 microgram/ml, the antiviral agent acyclovir did not depress selected cellular immune responses that are important for successful elimination of invading herpes group viruses.

Cellular cooperation in mitogen-induced human leukocyte inhibitory factor (LIF) synthesis

Interactions between human T and B lymphocytes and between lymphocyte subpopulations and accessory cells in lymphokine synthesis were investigated. The cells were stimulated with leukoagglutinin (LA), concanavalin A (Con A), protein A (prot A) and anti- β 2-microglobulin (anti- β 2m). The presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose-migration method. The results indicated that monocytes augmented LIF synthesis of T cells but suppressed that of B cells. Monocyte-helper effect was mediated by both cell-cell contact and soluble factors. In addition, T lymphocytes were found to augment B-cell LIF production. B lymphocytes enhanced Con A- but suppressed LA-induced LIF production by T cells. T-cell/B-cell collaboration was based on a direct cell-cell contact and no soluble factors were found.

Suppression of leukocyte inhibitory factor (LIF) production and [ 3H]thymidine incorporation by concanavalin A-activated mononuclear cells

The capacity of human mononuclear (MN) cells pretreated with concanavalin A (Con A) to suppress the activity of fresh phytohemagglutinin (PHA)-pulsed mononuclear cells was assessed. Con A-pretreated MN cells suppressed leukocyte inhibitory factor (LIF) activity in supernatants of PHA-pulsed cell cultures and [ 3H]thymidine incorporation by these cells. Suppression was obtained in both allogeneic and autologous systems with mitomycin-treated, irradiated, or untreated Con A-induced cells. Lymphocytes from two patients that, following treatment with Con A, did not suppress mitogen-induced proliferative response of normal cells also did not suppress LIF production.

Conditions for generating and assaying human leukocyte inhibitory factor (LIF) induced by purified protein derivative (PPD)

This report examines a variety of experimental conditions for the production of human leukocyte inhibitory factor (LIF). The data indicate that the production of LIF as measured in the indirect capillary-tube migration inhibition assay using human polymorphonuclear indicator cells accurately reflects delayed hypersensitivity to purified protein derivative (PPD) as detected by skin-test reactivity. Mononuclear cells and semipurified lymphocytes separated from whole blood by sedimentation in Ficoll-Hypaque were able to generate LIF in response to PPD. The quantity of cells responsible for LIF production were standardized and as little as 1 × 106 mononuclear cells were required for detectable LIF production. LIF produced by mononuclear cells required only temporary exposure to PPD, thus eliminating the necessity for a control to which antigen was added at the end of culture to antigen-free supernatants. In addition, the removal of antigen after a brief exposure helps to avoid the possible toxic effect of some antigens on the migrating polymorphonuclear leukocytes (PMNL) population. LIF production did not require extraneous serum protein (i.e., fetal bovine serum). Further, kinetic studies indicated t that LIF was detected as early as 8 hr following a 2 hr-exposure to PPD and that even a 1 hr-exposure was sufficient to generate measurable detectable quantities of LIF. After 48 hr of culture, supernatants were found to contain considerable amounts of LIF, with reactivity at dilutions as high as 1:40.

Cellular Sensitivity to Collagen in Rheumatoid Arthritis

We examined patients with rheumatoid arthritis for cellular sansitivity to native human Types I, II and III collagens. Mononuclear cells from 50 patients with rheumatoid arthritis, 21 with other inflammatory arthritides, 20 with osteoarthritis and 20 normal subjects were evaluated for the in vitro production of leukocyte inhibitory factor in response to collagen and a control antigen, streptokinase-streptodornase. By this assay, cells from 37 (74 per cent) and 39 (78 per cent) of the patients with rheumatoid arthritis responded to Types II and III collagens, respectively. In contrast, cells from the 41 patients with other kinds of arthritis and the normal group did not produce this lymphokine to collagens. There was no response to Type I collagen or to denatured alpha chains of these collagens. All four groups responded equivalently to streptokinase-streptodornase. These data demonstrate that most patients with rheumatoid arthritis exhibit cellular sensitivity to Types II and III collagens.

Production of leukocyte inhibitory factor (LIF) in Hodgkin's disease: Spontaneous production of an inhibitor of normal lymphocyte transformation

Skin reactivity and phytohemagglutinin (PHA)-induced lymphokine production (leukocyte inhibitory factor, LIF) were assessed in 30 patients with untreated Hodgkin's disease and found to be markedly depressed. These two parameters of cell-mediated immunity showed a 78.5% positive correlation. In addition increased LIF-like activity was found in supernatants of unstimulated mononuclear cells in 40% of Hodgkin's disease patients but rarely in other malignancies. A close association was found between spontaneous release of a LIF-like substance and depressed PHA-induced LIF production. Furthermore lymphocytes from 10 of 12 Hodgkin's disease patients studied demonstrated spontaneous uptake of [ 3H]thymidine. The supernatants produced by some unstimulated Hodgkin's disease mononuclear cells were able to suppress the lympho-proliferative response of normal cells to PHA. It is postulated that cellular unresponsiveness observed in Hodgkin's disease is partly explained by the production of an inhibitory factor (or factors), possibly by suppressor T cells.