Effect of L-asparaginase and hydrocortisone on human lymphocyte transformation and production of a mononuclear leucocyte chemotactic factor in vitro

Human peripheral lymphocytes stimulated in vitro with PHA produce a soluble factor which is chemotactic for homologous monocytes. The synthesis of this factor was found to precede the blastogenic response as measured by [³H] thymidine incorporation. In cultures of unseparated leucocytes the maximum of chemotactic activity was detected after 24 hr, whereas in supernatants from purified lymphocyte suspensions the maximal synthesis occurs after 72 hr. High doses of L-asparaginase from E. coli which have been found to prevent lymphocyte transformation completely have no influence on the production of the chemotactic factor. Therefore it seems possible that the induction of DNA synthesis by PHA and its effect on the production of a chemotactic factor depend on different biochemical mechanisms. In contrast hydrocortisone leads to a dose-dependent inhibition of both DNA synthesis and chemotactic response.     

Lactoferrin stimulates the production of leucocyte migration inhibitory factor by human peripheral mononuclear leucocytes.

The effect of lactoferrin on the migration of human polymorphonuclear cells was investigated. High concentrations of lactoferrin (greater than 250 micrograms/ml) markedly inhibited the migration of granulocytes under agarose. This migration inhibition could not be neutralized by an antibody against leucocyte migration inhibitory factor (LIF), suggesting a direct effect of lactoferrin on the granulocytes. Low concentrations of lactoferrin were, however, indirectly capable of inhibiting neutrophil migration. The overnight culture of mononuclear leucocytes with low concentrations of lactoferrin (10 micrograms/ml) resulted in the release of granulocyte migration inhibiting factors in the cell free culture supernatant. Strong evidence indicating that the migration inhibiting factors were due to LIF, was obtained in experiments whereby the inhibitory activity could be completely neutralized by anti-LIF antibodies. The lactoferrin-mediated stimulation of LIF release by mononuclear leucocytes could be neutralized by an anti-lactoferrin serum, but not by an anti-albumin serum, whereas PPD-induced LIF release was not affected by either antiserum. These findings suggest that lactoferrin besides its well known anti-microbial properties, may also play a regulatory role in the migratory response of polymorphonuclear cells during inflammatory conditions.     

Defective T-lymphocyte chemotactic factor production in patients with established malignancy

Lymphocytes from 22 patients with established malignancy were stimulated with concanavalin A (Con A), and supernatants were tested for T-lymphocyte chemotactic factor (LCF). LCF activity was measured using a leading front chemotaxis assay with normal human T cells as responders. Fifteen of the 22 patients tested produced LCF at a level of less than 2 standard deviation below the mean of control cells. In 10 patients where mononuclear cells were stimulated with Con A for 24, 48, and 72 hr, LCF activity was significantly reduced at all three time points averaging 38, 14, and 43% of control levels, respectively. In 13 of the 31 patients, patient T-cell migration in response to casein was measured and compared to the production of LCF by mononuclear cells from these same patients. A significant correlation was observed indicating that both the response of T cells to a migration stimulus, and the production of T-cell-derived LCF was comparably suppressed. The reduction in LCF production by mononuclear cells from patients with established malignancy was not reversed by the addition of indomethacin to the culture system during Con A stimulation indicating that inhibition was not mediated by excessive prostaglandin production. The addition of patient mononuclear cells or T cells to normal mononuclear cells resulted in the inhibition of normal cell LCF by patient mononuclear cells or T cells. This could not be attributed to the production of a lymphocyte chemotactic inhibitor by patient cells, but appeared instead to be due to the direct inhibition of normal cell LCF synthesis or release by patient mononuclear cells or T cells. Separation of patient T cells into Leu 2 suppressor/cytotoxic or Leu 3 helper/inducer T cells showed that the inhibitory activity was associated with the Leu-2 T-cell subset. Heterologous normal Leu 2 T cells did not suppress normal mononuclear cell LCF production suggesting that patient Leu 2 T cells were functionally activated as compared to normal Leu 2 cells. The decreased production of LCF coupled with a depressed T-cell migratory activity in patients with established malignancy may in part be responsible for suppressed cellular immune reactions in these patients and possibly the impairment of tumor rejection.     

Lymphocyte transformation in patients with amyloidosis

Lymphocyte transformation (measured by ¹⁴C-thymidine uptake) was studied in sixteen subjects, seven of whom had systemic amyloidosis. Impaired lymphocyte transformation was observed with Herpes simplex antigen, but not with purified protein derivative, phytohaemagglutinin and Candida in leucocyte cultures of patients with amyloidosis. A selective impairment of cell mediated immunity may, therefore, exist. Lymphocyte transformation was stimulated by a crude extract of amyloid fibrils in four out of seven patients with amyloidosis, but also in three of eight control subjects. This reaction cannot yet be used as diagnostic test for amyloidosis.     

Lymphocyte transformation and interferon production in human mononuclear cell microcultures for assay of cellular immunity to herpes simplex virus.

Interferon production and transformation in response to herpes simplex virus antigen were studied in microcultures of human mononuclear cells. Mononuclear cells consisting of monocytes and both T and B lymphocytes were purified by Ficoll-Hypaque gradients. Lymphocytes, predominantly T with 5% B, were obtained by passage of buffy-coat cells through nylon fiber columns. For some experiments, autochthonous macrophages and column-purified lymphocytes were stimulated with herpesvirus antigen. The effect of specific antibody and cell concentration on reactivity is described. Crude and purified antigens were compared as cell culture stimulants. Significant differences in transformation and interferon were observed between donors with a history of herpes labialis and donors with no detectable antibody, both in cultures prepared by Ficoll-Hypaque gradients and by column purification of lymphocytes. Cultures from seronegative donors prepared by Ficoll-Hypaque gradients produced interferon but did not transform when stimulated by herpes simplex antigen. "Immune" interferon production, that is, type II as opposed to type I, occurred only with autochthonous macrophage and column-purified lymphocyte cultures. Interferon produced by Ficoll-Hypaque-purified mononuclear cultures was type I, and its production was unrelated to immune status. Similarly, column-purified lymphocytes responded to herpes simplex virus antigen with type I interferon if obtained from a seropositive donor.     

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.     

Lymphocyte transformation test with liver-specific protein and phytohaemagglutinin in patients with liver disease.

(1) A new antibody has been found by immunofluorescence which reacts with cardiac conducting tissue using ox heart false tendons. It was detected in eight out of ninety-three cases of idiopathic heart block (8-6%), in one out of twenty-two cases of secondary heart block (4-5%) and in seven of 165 normal controls (4.2%), in titres varying from 1:10 to 1:40. Previous authors had indicated that this tissue might contain unique antigens. (2) Sera reacting with type I fibres in skeletal muscle (red zebra) were found to be of two varieties, one of which stained conducting fibres diffusely while it gave minimal staining of cardiac muscle; the other reacting with myofibrils in Purkinje cells and heart. (3) Some sera with high titre smooth muscle antibodies (SMA) reacted with conducting tissue together with skeletal and cardiac muscle, suggesting that the four tissues have at least one antigen in common. (4) Known non-organ specific antibodies behaved as expected on beef conducting fibres: striational fluorescence of myasthenia gravis sera reacted with the same patterns on Purkinje myofibrils; AMA and ANA produced IFL in expected locations; ribosomal antibodies reacted strongly, while LKM and reticulin antibodies showed no reactivity. (5) Although the incidence of specific Purkinje fibre antibodies was not significantly raised in idiopathic heart block, the clinical associations suggest that some cases might be related to autoimmunity possibly involving cell-mediated mechanisms as in polymyositis.     

Novel neutrophil chemotactic factor derived from human peripheral blood mononuclear leucocytes.

Human mononuclear leucocytes isolated from the peripheral blood by centrifugation on Ficoll-Hypaque cushions and adherent on plastic petri dishes, produced a chemotactic factor that attracted human neutrophilic granulocytes to the same extent as did optimal concentrations of the complement split product C5a and the leukotriene B4. The active component eluted from a Sephadex G-50 gel filtration column as a single peak with an apparent molecular weight of 10,000. The chemotactic activity was resistant to reductive cleavage of disulfide bonds and heating at 100 degrees C for 30 min but was lost when reduction and heating were combined. Digestion with a proteolytic enzyme eliminated the attractive potential. The data suggest that this is a novel chemotactic peptide. It is conceivable that it has been seen previously and was mistaken for a lymphokine or interleukin 1.     

Participation of a histamine-Sepharose-adherent subpopulation of human mononuclear cells in the production of leucocyte migration inhibition factor (LIF) in healthy children.

The separation of mouse splenic T lymphocytes into distinct subpopulations by fractionation on histamine-rabbit serum albumin Sepharose (H-RSAS) columns has been described. The H-RSAS-adherent T cells have been attributed regulatory functions associated with B cell activity, T cell-mediated cytotoxicity and the secretion of mediators such as immuno-interferon. The possibility that H-RSAS-adherent T cells exert a similar regulatory effect on an in vitro parameter of T cell-mediated immunity was investigated by assaying the production of leucocyte migration inhibition factor (LIF) in human blood samples, using the agarose droplet method. Phytohaemagglutinin (PHA) and BCG-purified protein derivative (PPD) were used as stimulants of LIF secretion which was measured as a percentage of inhibition of linear leucocytic migration. In normal individuals a highly significant (P less than 0.001) decrease was demonstrated in the production of LIF by peripheral blood leucocytes depleted of H-RSAS-adherent cells. Migration inhibition dropped from 36 +/- 11.7% to 21.2 +/- 12.9% in eighteen cases tested with PHA and from 29.3 +/- 11.7% to 17.2 +/- 9.8% in twelve cases tested with PPD. These results suggest the existence of a lymphocytic subpopulation involved in LIF production which expresses histamine receptors.     

Lymphocyte subset patterns and cytokine production in Alzheimer's disease patients

To investigate the signs of inflammatory processes in Alzheimer's disease (AD), we examined peripheral blood mononuclear cells (PBMC) from 51 AD patients (29 with mild and 22 with moderately severe dementia) and 51 age-matched healthy controls (HC), using flow cytometry to analyse the absolute number and the percentage of T, B and NK cells. We also studied the surface expression of CD25, CD28, CD57, CD71, CD45RA and CD45RO markers on cells CD4+ and CD8+. In 30 AD patients and 20 HC the production of IL-2, IFN-gamma, IL-10 and TNF-alpha by PBMC after stimulation with [25-35], [1-40] and [1-16] beta-amyloid (betaA) fragments was also evaluated. A significant decrease in circulating B and CD8+CD28- cells, as well as an increase in CD8+ cells expressing CD71+ and CD28+, was observed in AD patients. A significant decrease in IL-10 production was also found after stimulation of PMBC with betaA [1-40]. The decreased IL-10 production was not related to disease severity. The observed imbalance of immune peripheral cell subpopulations and decreased IL-10 production point to a reduction of suppressor cell function in AD patients.     

Cellular collaboration in the production of human leucocyte migration inhibition factor.

The participation of cell subpopulations in the expression of leucocyte migration inhibition factor (LMIF) in response to Concanavalin A and Protein A was evaluated for cells isolated from the peripheral blood of five healthy subjects. LMIF activity could not be attributed to the function of T cells, B enriched cells, or monocytes acting alone, or to a combination of B enriched cells and monocytes. The LMIF response was the result of a collaborative event that occurred between T cells and B enriched cells, or between T cells and monocytes.     

Lymphocyte predominance Hodgkin''s disease: a reappraisal based upon histological and immunophenotypical findings in relapsing cases

The clinical, morphological and immunological findings in nine cases of relapsing lymphocyte predominance Hodgkin's disease (LPHD) are examined. Six patients had initial biopsies demonstrating nodular lymphocytic and/or histiocytic (L&H) LPHD; Leu-M1 was not expressed by any of the atypical cells in these cases. All six demonstrated one or more recurrences of nodular L & H LPHD; four are currently free of disease, one died of non-Hodgkin's lymphoma and another died of leukaemia. Two patients had initial biopsies demonstrating diffuse LPHD, with only rare multilobated atypical cells (L & H variants). Both patients had recurrences interpreted as mixed cellularity Hodgkin's disease, 10 and 15 years after initial therapy and both died with lymphocyte depleted Hodgkin's disease. The atypical cells in the initial biopsies and in subsequent recurrences failed to express Leu-M1, but did express leukocyte common antigen. The initial biopsy from the final patient was histologically interpreted as focal involvement by LPHD, but interfollicular Hodgkin's disease was considered after the Leu-M1 stain revealed additional atypical cells. The disease relapsed and the patient died with typical nodular sclerosing Hodgkin's disease. The pattern of the relapses supports the concept that the histological entity of LPHD may include several distinct clinicopathological subgroups.     

Lymphocyte compartments in human spleen. An immunohistologic study in normal spleens and uninvolved spleens in Hodgkin''s disease.

A panel of monoclonal antibodies directed against T and B lymphocyte antigens was used to analyze the presence and localization of several lymphocyte subsets in 13 normal human spleens (3 of newborns) and 17 uninvolved spleens of patients with Hodgkin's disease. The distribution of cells in the white pulp corresponded with findings in other secondary lymphoid organs, except for the presence of a marginal zone, a unique compartment localized at the border of white and red pulp. The phenotype of the marginal zone cells indicates that it is likely that the marginal zone contains nonrecirculating as well as recirculating B cells, while T cells (of the T helper type) are also represented. Therefore, the notion that marginal zone cells are nonrecirculating IgM+, IgD- cells, appears to be an oversimplification. No clear differences were observed between spleens of patients with and without Hodgkin's disease.     

Defective in vitro antibody production to Varicella zoster and other virus antigens in patients with Hodgkin''s disease

Cultures of blood mononuclear cells stimulated with Varicella zoster antigen (VZA) produced specific anti-VZA antibody in 20 of 27 normal adults but only 10 of 47 patients with treated Hodgkin's disease. Serum anti-VZA antibody levels were the same in normals and patients. The deficiency of in vitro production was found in patients who had been off treatment for 6 or more years and was not related to splenectomy. Each of 11 untreated patients had absent in vitro production from blood cells but spleen cultures produced antibody readily. Using combinations of autologous spleen and blood cells, the defect was attributable to the blood B cells. Defective production was also found in response to influenza viruses and herpes simplex. We suggest that an abnormality in B cell circulation with localization of antibody producing B cells in spleen and lymph nodes, is a feature of treated and untreated Hodgkin's disease.     

核转录因子及炎性趋化因子在阿尔茨海默病患者脑组织中的改变

目的 探讨核转录因子(NF) -κBp65、炎性趋化因子单核细胞趋化蛋白1(MCP-1/CCL-2)、巨噬细胞炎性蛋白-1α(MIP-1 α/CCL-3)和胶质纤维酸性蛋白(GFAP)等在阿尔茨海默病(AD)发病中的改变。方法 选择尸体解剖后的10例AD患者及8例对照脑组织,用免疫组织化学EnVision方法检测海马、大脑额叶及颞叶皮质中NF-κBp65、MCP-1、MIP-1α、GFAP和Aβ1-42的表达。结果 与对照组相比,AD患者脑组织海马、额叶和颞叶皮质等部位均显示神经细胞中NF-κBp65[每高倍视野(×400)阳性细胞数分别为3.6±1.5、2.2±1.2和2.2±1.2]表达较对照组（每高倍视野阳性细胞数分别为0.31±0.20、0.25±0.20和0.25±0.20）明显增高(P＜0.05);MCP-1及MIP-1α在海马、额叶和颞叶皮质等部位神经细胞中（每高倍视野阳性细胞数分别为8.0±1.3、8.8±1.0、9.3±1.4;及8.1±1.5、12.5±1.1、6.4±1.1）表达均较对照组（每高倍视野阳性细胞数分别为4.5±0.9、4.5±0.6、4.0±1.8;及5.0±1.9、6.3±2.2、3.8±1.5）明显增多(P＜0.05);GFAP显示的星形胶质细胞数增多(P＜0.05),并大量聚集在老年斑周围。相关系数分析表明,在AD脑组织中MCP-1、MIP-1α蛋白水平与NF-κBp65的改变有关。结论 AD脑组织中核转录因子及炎性趋化因子表达升高,NF-κBp65可能调节MCP-1和MIP-1α的表达,其机制可能与Aβ引起的炎性作用有关。     

Alcoholic hepatitis: granulocyte chemotactic factor from Mallory body-stimulated human peripheral blood mononuclear cells

The characteristic histopathological features of acute alcoholic hepatitis include hyaline degeneration of hepatic parenchymal cells (Mallory bodies), hepatocellular necrosis, and granulocyte infiltration of the liver. The chemotactic response of neutrophils to highly purified Mallory bodies was studied. Mallory bodies, per se, were not chemotactic for granulocytes, nor did they generate chemotactic factors when incubated with serum. However, a factor(s) chemotactic for both granulocytes and mononuclear cells was generated when Mallory bodies were incubated with mononuclear cells, both from patients with alcoholic hepatitis or from normal controls. It was concluded that Mallory bodies stimulate peripheral blood mononuclear cells to release a factor chemotactic for granulocytes and mononuclear cells. This factor may be important in the etiology of the cellular infiltration in the livers of patients with alcoholic hepatitis.     

Immunohistochemical localisation of factor VIII-related antigen in Hodgkin''s disease

A series of 45 lymph nodes involved by Hodgkin's disease have been examined by means of the unlabelled antibody peroxidase-antiperoxidase (PAP) method for the demonstration of factor VIII-related antigen (F VIII R Ag). In addition, five lymph nodes showing reactive follicular hyperplasia were studied. In the specimens of nodular sclerosing Hodgkin's disease, many blood vessels were present, as demonstrated by virtue of F VIII R Ag in endothelial cells. These vessels were present within and, especially, around the nodules in this subtype; in addition the fibrous trabeculae were densely vascular. High vascularity was also a feature of specimens of the cellular variant of nodular sclerosing Hodgkin's disease. However, the other three Rye subtypes of the disease showed a striking paucity of F VIII R Ag-positive blood vessels. The reactive nodes showed numerous vessels around and between follicles but the lymph sinuses were consistently negative. Mast cells were present in all specimens but were especially frequent within and adjacent to the sinuses in reactive specimens; they were strongly positive for the F VIII R Ag, their staining being abolished by pre-adsorption of the primary antiserum with factor VIII itself.     

Ambroxol inhibits interleukin 1 and tumor necrosis factor production in human mononuclear cells

We have studied the effect of Ambroxol on the production of Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) in human mononuclear cells (MNC). For this purpose MNC were cultured for 24 h in the presence of endotoxin and different doses of Ambroxol. The results indicate that Ambroxol markedly inhibited IL-1 and TNF production at doses of 10–100 g/ml, without any apparent toxicity.     

Endotoxin-stimulated human macrophages produce a factor that induces polymorphonuclear leucocyte infiltration and is distinct from interleukin-1, tumour necrosis factor alpha and chemotactic factors.

Previously we reported that rabbit macrophages (M phi) in the presence of nanogram quantities of endotoxin (LPS) release factors that induce polymorphonuclear leucocyte (PMNL) infiltration into the skin of rabbits following i.d. injection. The predominant factor was a de novo synthesized protein of 45,000 MW on gel filtration that was distinguishable from IL-1 but not from TNF alpha. Here we examined human monocytes, in vitro monocyte-derived M phi and peritoneal M phi for the production of an analogous protein. Upon stimulation with LPS, they all rapidly (6 hr) produced a factor(s) that caused PMNL accumulation in the skin of rabbits when injected i.d. This activity, referred to as PMNL-recruiting activity (PRA), was heat labile and its production was blocked by cycloheximide. By Sephadex-G100 chromatography the major PRA of cultured M phi or peritoneal M phi had a molecular weight (MW) of 45,000-60,000. The active fractions were free of IL-1 (less than 0.2 U/ml) and Superose-12 FPLC chromatography separated the peak of PRA, which eluted at 45,000 MW, from TNF alpha, eluting at 20,000 MW. The peak PRA was not neutralized by antisera to IL-1 alpha, IL-1 beta, TNF alpha, IL-6 or GM-CSF, indicating that it was distinct from these cytokines. The major PRA did not induce the migration of PMNL in vitro in a filter chemotaxis assay. In contrast to the M phi, the major PRA produced by LPS-stimulated monocytes eluted at 15,000-20,000 MW, contained IL-1 activity and was neutralized by antisera to IL-1 alpha and IL-1 beta. Monocytes from a few donors also produced the 45,000-60,000 MW PRA simultaneously. We conclude that human peritoneal M phi and in vitro monocyte-derived M phi exposed to LPS secrete a protein of 45,000-60,000 MW, which is a potent inducer of PMNL infiltration but is distinct from IL-1, TNF alpha, IL-6, GM-CSF and PMNL chemotactic factors.