Transferencia in vitro de inmunidad a PPD con extracto dializable de leucocitos de calostro humano / In vitro immunity transference of PPD with human dialysable colostral leukocyte extract

Se pretende demostrar la transferencia de hipersensibilidad a PPD en un modelo in vitro con extracto dializable de leucocitos de calostro de madres PPD- y PPD+ (EDLC PPD- Y PPD-), a través de la medición de la actividad del factor inhibidor de la migración de leucocitos (LIF) de sangre de cordón de recién nacidos de madre PPD+. En los resultados se observa que el EDLC PPD+ incubado con leucocitos de recién nacidos de madres PPD- tuvieron inhibición de la migración de los leucocitos, comprados con la migración de los leucocitos incubados con EDLC PPD-; lo que sugiere que en el modelo in vitro se transfiere hipersensibilidad a PPD con el EDLC.

[Monocyte locomotion inhibiting factor (MLIF) produced by E. histolytica induces an increase of cAMP in human monocytes]. / El factor inhibidor de la locomoción de monocitos (FILM) producido por E. histolytica induce aumento del AMPc en los monocitos humanos.

The supernatant fluid of axenically grown E. histolytica contains a factor (MLIF) which inhibits the locomotion of human monocytes (including chemotaxis) without affecting that of human polymorphonuclear leucocytes. Locomotion, like other cellular functions, is modulated by changes in intracellular cAMP and cGMP. The consensus--with some exceptions--is that while rises in cGMP accompany locomotion, an increase in cAMP (without a concomitant fall in -cGMP) occurs with inhibition of cellular movement. We measured by radioimmunoassay the cAMP concentration of human monocytes exposed to inhibitory concentrations of MLIF. A significant (p less than 0.005) rise in monocyte cAMP was found, comparable to that observed with the use of forskolin, a well known cAMP stimulator. The control studies using plain axenic medium, not only failed to reveal any rise in cAMP but disclosed a small, yet not significant drop in intracellular cAMP. These results suggest that MLIF (like other locomotion inhibitors, i.e. prostaglandins E1, A1 and isoproterenol) produces a significant increase in intracellular monocyte cAMP. This modification in intracellular signals may contribute to the inhibition in monocyte locomotion, an event during which an increase in pericentriolar microtubules has also been observed.

Regulation of lymphocyte motility by macrophages: characterization of a lymphocyte migration inhibitory factor derived from a macrophage-like cell line.

An inhibitory factor on lymphocyte migration was detected using a capillary random migration assay in the culture supernatant of peritoneal exudate macrophages cultured at concentrations greater than 8 x 10(6) cells/ml. After examining different macrophage-like cell lines, J774A.1 cells were found to produce this inhibitory factor, which was termed lymphocyte migration inhibitory factor (LMIF). The inhibitory effect of LMIF on the migration of spleen lymphocytes, thymocytes, and bone marrow cells was determined. The migration of thymocytes was more sensitive to LMIF than was the migration of spleen lymphocytes and bone marrow cells. Interestingly, when the effect of LMIF was tested on the migration of spleen T cells and B cells, T cells were more sensitive than B cells. When the thymocytes were separated by peanut agglutinin into mature and immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes to the effect of LMIF, suggesting that the greatest effect of LMIF was on the migration of mature T cells. Partial purification of LMIF by ion-exchange and gel-filtration chromatography revealed that it is approximately 14,000 in molecular weight and could exist in either monomeric or dimeric forms. The possible role of this factor in an immune response is discussed.

Cell-mediated immune responses to artificial food additives in chronic urticaria.

In some cases of chronic urticaria it is suspected that food additives such as tartrazine and sodium benzoate or salicylates may play a role in the pathogenesis of the condition. Since, at times, chronic urticaria may appear histologically similar to a mild cell-mediated immune response, the release of the T cell-derived lymphokine leucocyte inhibitory factor (LIF), in response to incubation with these additives and with acetylsalicylic acid (ASA), was measured in vitro using cells from normal controls, from patients with chronic urticaria with or without clinically associated additive sensitivity and from patients with asthma with or without associated ASA sensitivity. It was found that significant production of LIF occurred in response to tartrazine and sodium benzoate in those individuals with chronic additive induced urticaria. In addition, tartrazine caused LIF release from mononuclear cells of ASA-sensitive asthmatics. These results may indicate a possible role for additive-induced cell-mediated immune responses in the pathogenesis of some cases of chronic urticaria and suggest a potential diagnostic test for this condition.

Studies on migration inhibitory factors of non-lymphoid origin.

A large number of mouse fibrosarcoma and adult guinea pig fibroblast cultures were examined for their ability to produce migration inhibitory activity. In most cases culture supernatants were found to inhibit macrophage migration, in a dose-dependent manner. Toxicity of the tested material could be excluded by: a) experiments using colchicine as a stimulator of macrophage migration and, b) examination of the effect of test materials on macrophage monolayer cultures. Additionally, migration inhibitory activity was found in fibroblast, but not fibrosarcoma frozen and thawed extracts. Furthermore, incubation of cells with puromycin could only inhibit production by fibrosarcoma, thus suggesting that the fibroblast activity was due to preformed cellular constituents. Fractionation of concentrated culture supernatants by Sephadex G-200 gel filtration showed that the activity derived from fibrosarcoma cells could be eluted in a narrow molecular weight fraction (18,000-22,500), whereas the fibroblast activity was heterogenously distributed over a wide range. Migration inhibitory activity in fibroblast extracts was mainly associated with higher molecular weight material. Differences could be demonstrated between these activities and lymphocyte migration inhibitory factor, including inhibition by methyl-pentoses and the presence of macrophage aggregating activity.

Partial purification and physicochemical properties of human T cell migration inhibitory factor (TIF).

Supernatants from 24 hr cultures of PHA-pulsed human T lymphocytes inhibit the migration of human peripheral blood T lymphocytes and guinea pig macrophages in vitro. The factor responsible for the inhibition of T lymphocytes provisionally called TIF (T cell migration inhibitory factor) was separated from MIF by preparative PAGE, had apparent molecular weight (m.w.) of 1,000-10,000 daltons and isoelectric point of 3.1. TIF activity was resistant to treatment with trypsin, chymotrypsin and neuraminidase but sensitive to PMSF (phenyl-methyl-sulfonyl-fluoride). This suggests that TIF is presumably different from human MIF and may represent a novel lymphokine which preferentially affects T cell migration in vitro.

Functional characteristics of histamine receptor-bearing mononuclear cells. II. Identification and characterization of two histamine-induced human lymphokines that inhibit lymphocyte migration.

Although functional histamine receptors have generally been restricted to those human T lymphocytes expressing suppressor cell functions, more recent evidence suggests that histamine receptor-bearing human T lymphocytes are functionally heterogeneous and capable of other immunomodulatory activities. Lymphocyte chemoattractant factor (LCF) is a cationic sialoprotein with an apparent m.w. of 56,000, whose production is limited to histamine-type 2 receptor-bearing human T cells. LCF is selectively chemokinetic for T lymphocytes, and presumably contributes to the recruitment of unsensitized effector lymphocytes at inflammatory sites. In addition to LCF, Sephadex G-100 gel filtration of histamine-induced lymphocyte supernatants revealed two regions of migration inhibitory activity for human blood T and rat splenic lymphocytes. These regions corresponded to m.w. of 70,000 to 80,000 (LyMIF75K) and 30,000 to 40,000 (LyMIF35K). LyMIF75K had a single pI of 7.5 to 8.0, and its biologic activity was sensitive to trypsin but not to neuraminidase or heat (56 degrees C). LyMIF35K had a single pI of 8.5 to 8.8, and its biologic activity was sensitive to neuraminidase and heat but not to trypsin. These LyMIFs therefore appeared to be distinct from one another and physicochemically different from other migration inhibitory lymphokines. All three lymphokine activities appeared within 4 hr of incubation. The minimum concentration of histamine required to stimulate production of the LyMIF was 10(-6) M. Lymphocytes that did not adhere to a histamine affinity matrix were unable to produce either LyMIF upon subsequent stimulation with histamine or concanavalin A (Con A). Lymphocytes incubated with histamine and diphenhydramine produced LCF but neither LyMIF, whereas cells incubated with histamine in the presence of cimetidine produced both LyMIF but not LCF. These data suggest that a subset of lymphocytes defined by the presence of histamine-type 1 receptors are capable of producing two distinct species of lymphocyte migration inhibitory activity. These cells may contribute to the immobilization of effector T lymphocytes chemokinetically attracted to certain inflammatory sites.

Antigen-independent leukocyte random locomotion inhibitory activity in human ultrafiltrated leukocyte extracts.

Human ultrafiltrated leukocyte extracts (MW less than 5000) were fractionated by Sephadex G-10 column chromatography and the effects of these fractions on leukocyte random locomotion were investigated in vitro. Fr-4, one of these fractions, had significant leukocyte random locomotion inhibitory activity, independent of the presence of mononuclear leukocytes. This inhibitory activity was not due to cytotoxic effects on leukocytes. As seen by scanning electron microscopy, the number of cell surface pseudopods on leukocytes incubated with Fr-4 was reduced. Fr-4A, one of three fractions separated from Fr-4 by Sephadex G-25 column chromatography, significantly inhibited leukocyte random locomotion. Fr-4A contained numerous components, one of which was identified as 2-deoxyribose, on the basis of thin-layer chromatography. Biologically 2-deoxyribose showed an inhibitory effect on leukocyte locomotion and a reduction of the extrusion of pseudopods on the surface of leukocytes, at the range of assayed concentrations. This inhibitory activity is probably derived from 2-deoxyribose.

Modulation of lymphocyte migration by human lymphokines. III. Characterization of a lymphocyte migration inhibitory factor (LyMIF35K).

The lymphokine that augments the migration of nonsensitized T lymphocytes (LCF) has been observed to be predominantly a chemokinetic factor, suggesting that separate lymphocyte migration inhibitory lymphokine(s) might exist. Utilizing a modified Boyden chamber assay, lymphocyte migration inhibitory activity was identified in the culture supernatants of human nylon wool-nonadherent blood mononuclear cells stimulated with concanavalin A in vitro for 48 hr. Sephadex G-100 gel filtration chromatography of these culture supernatants was shown to contain two regions of noncytotoxic migration inhibitory activity for nonsensitized human blood lymphocytes and rat splenic lymphocytes. The 30-40,000 dalton inhibitory activity was further characterized and noted to be cationic by ion-exchange chromatography and isoelectric focusing (pI = 8.6). Its biologic activity was sensitive to neuraminidase and to heat treatment but not to trypsin. The migration inhibitory activity of this factor (LyMIF35K) was directly proportional to its ability to increase lymphocyte adherence.

Physiochemical characterization of lymphocyte inhibitory factor (LyIF) isolated from avian B and T cells.

Thymic (T) or bursal (B) lymphocytes from chicks sensitized to Mycobacterium tuberculosis produce an avian lymphocyte inhibitory factor (LyIF). The physiochemical properties of both T and B LyIF were established by ultrafiltration which yielded four fractions with molecular weight ranges of greater than 100,000; 50,000-100,000; 10,000-50,000; and less than 10,000; enzymatic treatment with chymotrypsin and neuraminidase; varying pH; and heat exposure. These studies demonstrated that the maximum activity for both T and B LyIF was within a molecular weight range of 10,000-50,000. Both were sensitive to chymotrypsin and neuraminidase treatment. Both were stable at 56 degrees C for 30 min and resistant to changes in pH from 5 to 9. T-Cell migration was inhibited equally by B or T LyIF, while B-cell migration was inhibited to a lesser extent by T LyIF and B LyIF. Further experiments should establish the reasons for these observed differences in cross-reactivity.

In vitro induction of chronic myeloid leukemia associated immune reactivity in normal human lymphocytes by xenogeneic immune RNA.

Peripheral blood lymphocytes from normal human donors were treated with immune RNA (IRNA) prepared from lymphoid tissues of guinea pigs immunized with chronic myeloid leukemia (CML) cells, normal leukocytes (WBCs) and normal bone marrow (BM) cells, in order to study whether IRNA can confer specific immunoreactivity on normal lymphocytes. The IRNA treated lymphocytes were further exposed to solubilized membrane antigens from CML cells, normal WBCs and BM cells in a criss-cross fashion. The migration inhibition factor produced by these lymphocytes was tested in an in vitro leukocyte migration inhibition assay using normal leukocytes. The results indicated that IRNA prepared from guinea pigs immunized with all the three cell types could transfer the immune reactivity to normal cells in response to the respective antigens. The WBC IRNA incubated lymphocytes did not react with BM cell and CML cell antigens. A potent transfer of specific reactivity was shown by CML IRNA. IRNA produced against BM cells and CML cells demonstrated considerable cross reactivity perhaps due to shared immature cell antigens.

Migration inhibition caused by EBV-specific 48K subcomponent of EBNA and the associated 53K cellular protein.

Leukocytes from EBV-seropositive but not seronegative healthy donors responded with significant migration inhibition to the 48K subcomponent of the Epstein-Barr virus determined nuclear antigen (EBNA), known to carry the virally determined antigenic specificity. A concentration of 10 micrograms/ml was still effective while 5 micrograms/ml had no detectable effect. EBNA-associated cellular 53K protein had no effect by itself, but it potentiated the effect of 48K, even if the latter was added at the subliminal concentration of 5 micrograms/ml. The related 53K protein, isolated from EBV-negative human lymphoma cells, was also effective, whereas the corresponding murine-tumor-associated 53K had no potentiating effect. Immunization of mice with an extract of DNA-binding proteins from EBV-carrying Raji cells, known to contain both 48K and 53K, induced a significant macrophage migration inhibition response, to both human 48K and 53K. Murine 53K was ineffective, however. Human but not murine 53K increased the migration inhibitory activity of subliminal concentrations of 48K in the murine macrophage system as well. These findings suggest that human but not murine 53K may reconstitute with 48K (EBNA) to form a highly immunogenic complex.