Physicochemical characterization of human fibroblast migration inhibitory factor

We recently reported a new lymphokine activity that affects fibroblasts by inhibiting their spontaneous migration. Human fibroblast migration inhibitory factor (FIF) obtained from concanavalin A (Con A)-stimulated human lymphocytes was characterized by Sephadex gel filtration and by enzyme treatment. FIF was found to be stable at 56 degrees C for 15 min but destroyed at 80 degrees C or at pH lower than 5. Gel filtration revealed two peaks of FIF activity 15,000 and at 34,000 Da. FIF activity was lost following treatment with trypsin, chymotrypsin, and neuraminidase and FIF could not be generated in the presence of inhibitors of glycosylation, suggesting that the molecule was a glycoprotein. FIF could be removed by adsorption to human fibroblasts but not to PMN, monocytes, or red blood cells. Further studies were carried out to investigate the role of sugars in the interaction of FIF with the target cells. Human FIF activity was significantly reduced in the presence of several sugars including alpha-methyl-D-mannoside, L-xylose, N-acetyl-D-glucosamine, D-mannose, L-rhamnose but not L-fucose. Preincubation of human fibroblasts with alpha-methyl-D-mannoside prevented their response to FIF. In contrast, pretreatment of fibroblasts with mannosidase had no effect, suggesting that alpha-methyl-D-mannoside was an essential component of the FIF molecule recognized by the FIF receptor on fibroblasts.     

Quantitative assessment of the adherence of normal human blood mononuclear cells to vascular endothelial cell monolayers

We have developed a method to reliably quantitate the in vitro adherence of ⁵¹Cr-labeled blood mononuclear leukocytes to cultured monolayers of vascular endothelial cells from human umbilical veins. Normal mononuclear leukocytes adhered to endothelial cells more than to cover glass at all studied time periods over 4 hr with major differences seen at 2 hr (9.7 ± 1.2% vs 3.7 ± 1.1%; P < 0.01). Only a minority of cells adhering to endothelium were esterase positive. Similar patterns of binding were seen using varying concentrations of suspended mononuclear cells (1–4 × 10⁶/ml) simulating that occurring in vivo in different clinical states. This approach shows promise for in vitro approaches to lymphocyte-vascular endothelial interactions in human immune/inflammatory disorders.     

Regulation of growth of human diploid fibrobalasts by factors elaborated by activated lymphoid cells

Fibroblast growth and synthesis activities appear to be under exquisite control. This control is mediated in part by substances present in blood plasma or released by other cells. We have studied the role of peripheral blood mononuclear cells (PBM) activated with phytohemagglutinin-P (PHA) on DNA synthesis, proliferation, and the cell cycle of human diploid fibroblasts. Culture medium from activated but not from unactivated PBM cultures inhibited fibroblast DNA synthesis and growth in a dose-dependent manner. The activity, which was designated as lymphocyte factor (LF), was very potent; it inhibited 50% of the DNA synthesis and cell growth at a dilution of 1:160. It has a molecular weight between 50,000 and 100,000 daltons and it is destroyed by trypsin digestion or by heating at 80°C for 30 minutes. The activity was not due to the presence of prostaglandin. Furthermore, using immunoprecipitation and affinity chromatography, it was shown conclusively to to be distinctly different from alpha lymphotoxin (α-LT). It was not cytotoxic, as shown by the ⁵¹chromium release technique. Using flow microfluorimetry it was shown that the activity regulates fibroblast growth by preventing quiescent cells in the G⁰ or G¹ stage of the cell cycle from entering the S phase. Cells already in S at the time of exposure complete DNA Synthesis but cannot divide, and they accumulate in G². The activity also has marked effects on protein synthesis. Activated mononuclear cells may play a major role in regulating fibroblast growth and synthesis in normally healing wounds and in acute and chronic inflammatory processes.     

Binding and thermal dissociation of nerve growth factor and its receptor on human melanoma cells.

The interaction between mouse nerve growth factor (NGF) and its receptor was studied on live and formaldehyde-fixed human melanoma cells in culture. These cells contain 5–8 × 10⁵ NGF receptors per cell. The pH optima of this ligand-receptor association was 6.4. The kinetics of dissociation at 4°C was similar for the fixed and live cells; at 22°C, NGF readily dissociated from the fixed cells whereas the live cells showed little dissociation. Radioactive NGF which had been dissociated at 4°C from NGF-receptor containing cells was able to rebind with greater efficiency. With the dissociation of NGF from the cell surface, there was a concomitant increase in the number of available receptor sites. The initial events in the interaction of NGF and its receptor on human melanoma cells are reversible.     

Cultured cells from renal cortex of hibernators and nonhibernators. Regulation of cell K+ at low temperature

Cells were grown as primary monolayer cultures from kidney cortex of guinea pigs (nonhibernators), hamsters and ground squirrels (both hibernating species). When plates of cells were placed at 5 °C, cells of guinea pigs lost 37% of their K⁺ in 2 h and those of the hibernator lost about 10%.Uptake of ⁴²K into the cells exhibited a simple, single exponential time course at both temperatures. Unidirectional efflux of K⁺ was equal to K⁺ influx in all cultures at 37 °C and, within limits of error, in hibernator cells at 5 °C. Efflux was 3- to 5-fold greater than influx in guinea pig cells at 5 °C.After 2 h in the cold the ouabain-sensitive K⁺ influx remaining (7–15% of that at 37 °C) was about the same in the cells of the 3 species. Cells from active hamsters and from hibernating ground squirrels, however, exhibited significantly greater pump activity after 45 min in the cold (19 and 14%, respectively). The stimulation of K⁺ influx by increasing [K⁺]_o did not show an increase in K_m⁺ at 5 °C in cells of guinea pigs and ground squirrels. Lowering [K⁺]_c and/or raising [Na⁺]_c by treatment in low- and high-K⁺ media caused only slight stimulation of K⁺ influx, except in cells of ground squirrels at 5 °C in which the stimulation was at least 11-times greater than at 37 °C or in cells of guinea pigs at either temperature.This altered kinetic response of K⁺ transport to cytoplasmic ion stimulation with cooling accounted for about one-third of the improved regulation of K⁺ at 5 °C in ground squirrel cells; the other two-thirds was attributable to a greater decrease in K⁺ leak with cooling. The inhibition of active transport by cold in all 3 species was much less severe than that previously seen in any (Na⁺ + K⁺)-ATPase of mammalian cells.     

Evidence for a surface receptor for human migration inhibitory factor on a B-lymphoid cell line.

Reception by PGLC-33H target cells for the migration inhibitory factor (MIF) produced by this established line has been investigated by pulse time and temperature dependence, MIF absorption, and abrogation by trypsinization. PGLC-33H supernatants containing MIF were concentrated 5× with Carbowax and dialyzed against serum free RPMI-1640 before use. Prior to standard capillary migration assay a minimum 30 min pulse of MIF at 37 °C is required for significant migration inhibition (MI > 20%). No significant MI is observed when cells are pulsed at 4 °C for up to 2 hr. Preincubation with PGLC-33H for 1 hr at 37 °C reduces activity of supernatants from 38 to 13% MI; at 4 °C to 27% MI. Trypsinization of target cells for 30 min at 25 °C abrogates response to MIF (43 to ?14% MI). Trypsinized cells did not reduce activity of supernatants. MIF activity is abolished (32 to 3% MI) in samples preincubated with supernatants of the trypsinized cells inactivated with serum. These data suggest that cells from the human B-lymphoid cell line PGLC-33H have a surface receptor for human MIF.     

Antigen responsiveness of an established human B-lymphoid cell line I. Evidence for a surface receptor for Candida antigen detected by direct migration inhibition

Responsiveness to a soluble crude Candida albicans antigen (CaAg) by a long-term human lymphoid cell line with B-cell characteristics (PGLC-33H) is demonstrated by the direct migration inhibition (DMI) assay. An inability to detect comparable CaAg responses in other tested B- and T-lymphoid cell lines suggests that this may be a specific response for PGLC-33H cells. CaAg responsiveness is completely abrogated by trypsinization of cells. Regeneration of CaAg responsiveness is temperature dependent, detectable in 8–14 hr in cells maintained at 37°C. Trypsinized cells kept at 4°C do not regain CaAg response. The rate of cell-CaAg interaction is rapid and not temperature dependent. Cells pulsed with CaAg at 37, 25, and 4°C demonstrate comparable DMI responses following a 2-min pulse with CaAg. These findings suggest that PGLC-33H cells have surface receptors for CaAg which result in the inhibition of cell migration upon exposure to antigen.     

The predominant contribution of platelets to baseline and ascorbate-stimulated increments in cyclic GMP in human mononuclear leukocyte preparations.

In nine consecutive experiments with Ficoll-Hypaque-purified human mononuclear leukocytes containing 2.8 (range 1.1–4.3) platelets per leukocyte, 2–5 mM sodium ascorbate produced a 14-fold (range, 7- to 18-fold) rise in guanosine 3′: 5′-cyclic monophosphate (cyclic GMP) from baseline levels of 0.103 ± 0.056 pmol/10⁷ mononuclear leukocytes. In five experiments with mononuclear leukocytes prepared by the Ficoll-Hypaque method from human blood depleted of platelets by defibrination, 2–5 mM sodium ascorbate produced a twofold (range, one- to fourfold) rise in cyclic GMP from baseline levels of 0.030 ± 0.012 pmol/10⁷ mononuclear leukocytes. Thus, platelets contribute substantially to baseline and ascorbate-stimulated levels of cyclic GMP in standard Ficoll-Hypaque preparations of mononuclear leukocytes. The rise in cyclic GMP concentration in mononuclear leukocyte preparations elicited by ascorbate was independent of a calcium requirement, persisted for up to 3 hr in the presence of ascorbate, and was prevented by the introduction of nonsteroidal anti-inflammatory agents such as aspirin and indomethacin (ID₅₀ = 105 and 23.5 μM, respectively).     

E-rosette formation at 37 °C: Analysis with monoclonal OKT antibodies

When cultured in the presence of PHA, a proportion of human peripheral blood mononuclear cells acquires the capacity to form E rosettes with sheep erythrocytes that are resistant to incubation at 37 °C. The nature of this 37 °C stable E-rosette formation was investigated using a panel of monoclonal OKT antibodies directed to human T-lymphocyte surface antigens. OKT11A antibody, at a concentration of 0.2–0.4 μg/ml, markedly blocked 37 °C E rosetting. OKT1, OKT3, OKT4, OKT6, and OKT8 antibodies, when tested at 10 μg/ml, show no such inhibiting activity. Quantitative studies with ¹²⁵I-labeled OKT11A indicated that the antibody interacted strongly with both 37 °C E-rosetting and nonrosetting cells, the association constant being 1.6–2.0 × 10⁹M^?1. However, on the average, a threefold higher concentration of OKT11A receptor sites was found on 37 °C E-rosette-forming cells (14.8 × 10⁴ sites/cell) than on nonrosetting cells (4.8 × 10⁴ sites/cell). Our data suggest that 37 °C E-rosette formation is governed by a lymphocyte surface determinant recognized by OKT11A antibody. “Overexpression” of OKT11A antigenic sites on a proportion of PHA-stimulated lymphocytes may explain their capacity to form 37 °C stable E-rosettes.     

Influence of lectins on the binding of 125I-labeled EGF to human fibroblasts.

Lectins that interact with mannose (concanavalin A), galactose (ricin, abrin), or N-acetylglucosamine (wheat germ agglutinin) block ¹²⁵I-labeled EGF binding to the surface of cultured human fibroblasts at 37° or 5°. Lectins specific for fucose or N-acetylgalactosamine, soybean agglutinin or gorse lectin, respectively, do not interfere with growth factor binding. The inhibition of ¹²⁵I-labeled EGF binding by concanavalin A at 37° or 5° could be reversed rapidly by the addition of α-methyl mannoside. The results suggest that the fibroblast membrane receptor for EGF is, or is closely associated with, a glycoprotein or glycolipid that contains mannose, galactose and N-acetylglucosamine residues.     

Plasminogen activator and MIF-like activities in Kirsten virus transformed mouse NIH culture fluids.

Kirsten virus transformed mouse NIH cells produce both a macrophage migration inhibition activity for guinea pig and mouse peritoneal exudate cells and a plasminogen activator. The migration inhibition factor activity exhibited thermal stability up to 80°C while the plasminogen activator was inactivated after 15 minutes at 70°C. Separation of these activities was achieved by absorption of the migration inhibition activity on agarose-fucosamine or high speed centrifugation.     

Amplification of the activity of human leukocyte inhibitory factor (LIF) by the generation of a low molecular weight inhibitor of PMN leukocyte chemotaxis

Leukocyte inhibitory factor (LIF), which was derived from human peripheral blood lymphocytes by stimulation with concanavalin A ad partially purified by Sephadex G-100 gel filtration, inhibited the in vitro spontaneous migration and chemotaxis of human PMN leukocytes as assessed in a Boyden chamber micropore filter assay. The inhibitory activity was attributed to LIF, a principle defined in terms of its inhibition of PMN leukocyte migration from glass capillary tubes since it was preferentially directed to PMN leukocytes as compared to mononuclear leukocytes, exhibited a size comparable to LIF by gel filtration, and was inactivated by diisopropyl fluorophosphate in parallel with LIF. Incubation of PMN leukocytes with LIF released additional inhibitory activity, distinct from LIF, which resembled the neutrophil-immobilizing factor (NIF) by virtue of its approximate m.w. of 4000 by filtration on Sephadex G-25, inactivation by trypsin digestion, and preferential noncytotoxic inhibition of spontaneous migration and chemotaxis of PMN leukocytes as compared to mononuclear leukocytes. Thus LIF inhibits PMN leukocyte migration both by a direct action on the cells and by an amplification pathway that is mediated by low m.w. chemotactic inhibitors similar to NIF.     

Cryopreservation of human granulocytes.

Granulocyte preservation was undertaken using hydroxyethylstarch for both sedimentation of red cells and cryopreservation of buffy coat white cells from CPD whole blood. Buffy coats were mixed with HES to a final concentration of 4% (w/v) and hematocrit of 30%, and sedimented in inverted plastic syringes. The leukocyte enriched (100–500×) supernatant was frozen at 2.0 °C/min to ?80 °C (and stored frozen up to 3 months). Alternatively, sedimented leukocytes were frozen after a slow addition of 10% DMSO to 5%. Tubes were thawed at 37 °C, and DMSO was removed by dilution with Hank's solution containing CPD and centrifugation. The pellets of granulocytes were resuspended in Normosol.Buffy coat from 10 units yielded 60 ± 9.7% of the available whole blood leukocytes, of which 43 ± 14% were recovered after sedimentation in HES. Freezing in DMSO yielded all, 101% of the prefrozen leukocytes. Postthawed viability of granulocytes was estimated morphologically and by their ability to inhibit the rate of growth of E. coli. Complete inhibition was observed at a ratio of one E. coli to one granulocyte. Postthawed granulocytes were characterized by high myeloperoxidase activity and exclusion of trypan blue. Approximately 25% of the total available granulocytes in CPD whole blood were recovered.     

Visualization of several binding sites for basic fibroblast growth factor (FGF-2) on fibroblasts by photoaffinity labeling: Evidence for intracellular complexes

The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azido-benzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37°C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Flg receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4°C with ¹²⁵I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170–180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130–160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with ¹²⁵I-HSAB-FGF-2 at 4°C and then washed, were shifted to 37°C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of ¹²⁵I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex. © 1996 Wiley-Liss, Inc.     

Differences in proteins secreted by human fibroblasts and muscle cells in culture

Cell cultures of human skin fibroblasts, myoblasts, and fused muscle cells were grown in the presence of [¹⁴C]leucine or a mixture of [¹⁴C]amino acids. The proteins synthesised and secreted or leaked into the culture medium during radio-labelling were separated by one and two-dimensional PAGE and detected by fluorography. Four major bands of M_r 54 kD, 52 kD, 51 kD, and 49 kD were present at greatly increased concentration in fibroblast media. These fibroblast-specific polypeptides can be readily detected in myoblast/fibroblast cocultures with fibroblast content as low as 5%.     

Copper stimulates proliferation of human endothelial cells under culture

Copper ions stimulate proliferation of human umbilical artery and vein endothelial cells but not human dermal fibroblasts or arterial smooth muscle cells. Incubation of human umbilical vein endothelial cells for 48 h with 500 μM CuSO₄ in a serum-free medium in the absence of exogenous growth factors results in a twofold increase in cell number, similar to the cell number increase induced by 20 ng/ml of basic fibroblast growth factor under the same conditions. Copper-induced proliferation of endothelial cells is not inhibited by 10% fetal bovine serum or by the presence of antibodies against a variety of angiogenic, growth, and chemotactic factors including angiogenin, fibroblast growth factors, epidermal growth factor, platelet-derived growth factor, tumor necrosis factor-α, transforming growth factor-β, macrophage/monocyte chemotactic and activating factor, and macrophage inflammatory protein-1α. Moreover, despite the previous observations that copper increased total specific binding of ¹²⁵I-angiogenin to endothelial cells, binding to the 170 kDa receptor is not changed; hence, the mitogenic activity of angiogenin is not altered by copper. Copper-induced proliferation, along with early reports that copper induces migration of endothelial cells, may suggest a possible mechanism for the involvement of copper in the process of angiogenesis. J. Cell. Biochem. 69:326–335, 1998. © 1998 Wiley-Liss, Inc.     

Heat stress disrupts intestinal stem cell migration and differentiation along the crypt–villus axis through FAK signaling

During heat stress (HS), the intestinal epithelium suffers damage due to imbalance of tissue homeostasis. However, the specific mechanism by which intestinal stem cells (ISCs) migrate and differentiate along the crypt–villus axis to heal lesions upon insult is unclear. In our study, C57BL/6 mice and IPEC-J2 cells were subjected to normal ambient conditions (25 °C for 7 days in vivo and 37 °C for 18 h in vitro) or 41 °C. The results showed that HS impaired intestinal morphology and barrier function. The numbers of ISCs (SOX9⁺ cells), mitotic cells (PCNA⁺ cells), and differentiated cells (Paneth cells marked by lysozyme, absorptive cells marked by Villin, goblet cells marked by Mucin2, enteroendocrine cells marked by Chromogranin A, and tuft cells marked by DCAMKL1) were reduced under high temperature. Importantly, BrdU incorporation confirmed the decreased migration ability of jejunal epithelial cells exposed to 41 °C. Furthermore, intestinal organoids (IOs) expanded from jejunal crypt cells in the HS group exhibited greater growth disadvantages. Mechanistically, the occurrence of these phenotypes was accompanied by FAK/paxillin/F-actin signaling disruption in the jejunum. The fact that the FAK agonist ZINC40099027 reversed the HS-triggered inhibition of IPEC-J2 cell differentiation and migration further confirmed the dominant role of FAK in response to high-temperature conditions. Overall, the present investigation is the first to reveal a major role of FAK/paxillin/F-actin signaling in HS-induced ISC migration and differentiation along the crypt–villus axis, which indicates a new therapeutic target for intestinal epithelial regeneration after heat injuries.     

Differential effects of ouabain on human cell-mediated cytotoxicity. I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets.

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed ⁵¹Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10^?4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.     

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVI Dingo (Canis familiaris dingo) and dog (Canis familiaris)

As part of a programme of comparative measurements of P _d (diffusional water permeability) the RBCs (red blood cells) from dingo (Canis familiaris dingo) and greyhound dog (Canis familiaris) were studied. The morphologies of the dingo and greyhound RBCs [examined by light and SEM (scanning electron microscopy)] were found to be very similar, with regard to aspect ratio and size; the mean diameters were estimated to be the same (～7.2 μm) for both dingo and greyhound RBCs. The water diffusional permeability was monitored by using an Mn²⁺‐doping ¹H NMR technique at 400 MHz. The P _d (cm/s) values of dingo and greyhound RBCs were similar: 6.5×10^?3 at 25°C, 7.5×10^?3 at 30°C, 10×10^?3 at 37°C and 11.5×10^?3 at 42°C. The inhibitory effect of a mercury‐containing SH (sulfhydryl)‐modifying reagent PCMBS (p‐chloromercuribenzene sulfonate) was investigated. The maximal inhibition of dingo and greyhound RBCs was reached in 15–30 min at 37°C with 2 mmol/l PCMBS. The values of maximal inhibition were in the range 72–74% when measured at 25°C and 30°C, and ～66% at 37°C. The lowest value of P _d (corresponding to the basal permeability to water) was ～2–3×10^?3 cm/s in the temperature range 25–37°C. The E _a,d (activation energy of water diffusion) was 25 kJ/mol for dingo RBC and 23 kJ/mol for greyhound RBCs. After incubation with PCMBS, the values of E _a,d increased, reaching 46–48 kJ/mol in the condition of maximal inhibition of water exchange. The electrophoretograms of membrane polypeptides of the dingo and greyhound RBCs were compared and seen to be very similar. We postulate that the RBC parameters reported in the present study are characteristic of all canine species and, in particular in the two cases presented here, these parameters have not been changed by the peculiar Australian habitat over the millennia (as in the case of the dingo) or over shorter time periods, decades or centuries (as in the case of the domestic greyhound).