Interleukin 1 and prostaglandin production by cells of the mononuclear phagocyte system isolated from mycobacterial granulomas

A study has been made of the activity of interleukin 1 (IL-1) and prostaglandins (PGs) in the culture supernatants from unstimulated and lipopolysaccharide (LPS)-stimulated mycobacteria-induced granuloma cells. Both epithelioid cells from bacillus Calmette-Guerin (BCG)-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas, separated on a fluorescence-activated cell sorter using monoclonal antibody specific to guinea pig macrophages, spontaneously secreted low levels of IL-1 (assayed by thymocyte comitogenic and fibroblast mitogenic activities) into culture supernatants. However, culture supernatants from LPS-stimulated epithelioid cells showed significantly higher IL-1 activity than those from unstimulated cells. In contrast, LPS stimulation of M. leprae granuloma macrophages failed to enhance IL-1 production. Nevertheless, IL-1 activity in the culture supernatants from stimulated mycobacterial granuloma cells of both types was much lower than that from LPS-stimulated peritoneal exudate macrophage culture supernatants. There was no detectable amount of prostaglandin E2 (PGE2) in the culture supernatants from both unstimulated and LPS-stimulated BCG- and M. leprae-induced granuloma cells in comparison to much higher levels of PGE2 produced by unstimulated (0.28-6.2 ng/ml) or LPS-stimulated (greater than 15 ng/ml) peritoneal exudate macrophages. However, BCG granuloma cells either secreted prostaglandin F2 alpha (PGF2 alpha) spontaneously or produced comparable levels of PGF2 alpha to those from peritoneal exudate macrophages on stimulation, while M. leprae granuloma macrophages produced much lower levels of PGF2 alpha.     

Induction of leukotriene production before antigen challenge enhances antibody affinity in genetically selected mice

Mice genetically selected for their incapacity to produce high-affinity antibody to protein antigens in adjuvant (nonmaturing (NM) mice) were treated with indomethacin, an inhibitor of the cyclooxygenase pathway of arachidonic acid metabolism. Pretreatment with indomethacin significantly enhanced the affinity of antibodies produced 21 days after immunization with human serum albumin (HSA). Blockage of the cyclooxygenase pathway in this way was shown to induce the production of leukotrienes via the lipoxygenase pathway. The production of leukotrienes may well be responsible for the enhanced antibody affinity, since blockage of the lipoxygenase pathway in addition to the cyclooxygenase pathway reversed the effect. In an attempt to elucidate the mechanisms involved, IL-1 production and Ia expression by macrophages were examined. Ia expression by peritoneal cells from untreated NM mice was significantly lower than that by their high-affinity-producing counterparts 3 days after immunization. Indomethacin pretreatment raised inducible Ia antigen levels on macrophages of NM mice to those seen on cells from untreated high-affinity mice. Indomethacin treatment alone induced the production of IL-1 by macrophages in NM mice. However, 3 days after immunization and the withdrawal of indomethacin in NM mice, IL-1 production was significantly lower than the response of NM mice given antigen alone, suggestive of the induction of a feedback mechanism. Thus indomethacin pretreatment results in a cascade of events in macrophages which produce a decrease in IL-1 production and an increase in inducible Ia expression 3 days after antigen challenge.     

Amplification of immune T lymphocyte function in situ: the identification of active components of the immunologic network during tumor rejection

Previous data had indicated that sarcoma-bearing mice receiving combination therapy consisting of a single i.p. injection of cytoxan (CY) and an i.v. injection of tumor-sensitized T cells (immune cells) rejected the neoplasm. The reaction was immunologically specific and dependent on donor T cells. This report is concerned with the hypothesis that the transfer of immune cells results in the amplification of T cell responses at the tumor site. Using C57BL/6J mice bearing the syngeneic rhabdomyosarcoma MCA/76-9, we show that 6 to 9 days after combination therapy those components usually associated with the immunologic network were present at the tumor site. Tumor-associated macrophages (TAM) and lymphocytes (TAL) were shown to produce IL 1 and IL 2, respectively. The TAM expressed Ir gene products (Ia) and were able to present the synthetic polymer GAT to specifically sensitized lymphocytes. In addition, it was demonstrated by in situ labeling with 3H-TdR that lymphocytes associated with the regressing tumors were proliferating. The peak incorporation occurred 7 days after therapy, 24 hr before a significant increase in the T cell content of the tumors. The data indicate that those facets of the immunologic network necessary for amplification were present at the site of rejection.     

Induction of macrophage Ia expression by lipopolysaccharide and Listeria monocytogenes in congenitally athymic nude mice

Experiments were performed to analyze the mechanism by which lipopolysaccharide (LPS) modulates the expression of Ia by murine peritoneal macrophages in vivo. We investigated the effect of LPS on Ia expression in T cell deficient mice by using the congenitally athymic nude mouse model. Injection (i.p) of LPS into athymic (nu/nu) mice resulted in a dramatic increase in the expression and biosynthesis of Ia by peritoneal macrophages 7 days after injection. The magnitude and kinetics of this induction were equivalent to increases observed after LPS injection of euthymic (nu/+) mice. Viable Listeria monocytogenes also increased Ia expression in athymic mice, but in contrast to the induction observed in euthymic mice at 3 and 7 days after injection, increased Ia expression was not seen until 7 days. Ia induction by either LPS or L. monocytogenes in athymic mice was not due to the presence or development of mature T cell function as defined by assays for T cell mitogenesis and interleukin 2 production. We conclude that increased macrophage Ia expression by LPS and L. monocytogenes in vivo can occur in the absence of mature functioning T cells.     

B-cell-derived interleukin-1 (IL-1)-like factor. II. Sources, effects, and biochemical properties

A variety of types of human B-cell lines were evaluated for their ability to produce interleukin 1 (IL-1)-like factors. All of the eight Epstein-Barr virus (EBV)-transformed B lymphocyte lines, three of four of the EBV+ lymphoma lines, only three of seven of the EBV- lymphoma lines, and none of the three tested myeloma lines secreted some IL-1 activity. The IL-1-like factor produced by the cell lines was detected on the basis of its thymocyte comitogenic and/or fibroblast proliferative activities. Injections of partially purified IL-1-like factor from one of the EBV-transformed B-lymphocyte lines also induced the appearance of an acute phase protein (haptoglobin) in the serum of C3H/HeJ mice. These biological activities are identical with those of monocyte-derived IL-1. Thymocyte comitogenic activity and fibroblast proliferation activity from one of the EBV-B cell line-derived IL-1-like activities were not dissociable by biochemical procedures, including HPLC gel filtration and HPLC anion-exchange chromatography. However, the IL-1-like factor from one of the EBV-B lymphocyte cell lines was larger in size (25 kDa) and more acidic (pI 5.5) than monocyte-derived IL-1.     

益气补肾方药对化疗荷瘤小鼠免疫功能的影响

目的　探讨益气补肾方药 (由金匮肾气方加味人参、黄芪组成 ,以下均称方药 ,TS)对环磷酰胺 (CY)处理的荷H2 2 瘤小鼠非特异免疫功能的影响。方法　用CY处理荷H2 2 瘤小鼠 ,建立免疫力低下动物模型。用乳酸脱氢酶 (LDH)释放法分别检测NK细胞、巨噬细胞 (M)的活性 ,用RT PCR法检测白细胞介素 12 (IL 12 )mRNA在脾脏细胞中的表达。结果　TS CY组小鼠吸光度A值为 1 332± 0 5 10 ,明显高于CY组小鼠 (P <0 0 1)。TS CY组小鼠A值为 1 12 9± 0 2 80 ,明显高于CY组小鼠 (P <0 0 1)。此方药能明显提高CY所致免疫力低下小鼠NK细胞、M的活性 ,增加IL 12在脾细胞中的表达。结论　该方药可以明显提高环磷酰胺所致免疫力低下荷瘤小鼠的非特异免疫功能。     

Somatic cell hybrids between macrophages from Bcgr and Bcgs mice: characterization of MHC class II expression

In order to gain a better understanding of the regulation of MHC class II expression related to the Bcg gene, we have produced macrophage-macrophage somatic cell hybrids by fusing the RAW 309 macrophage cell line derived from BALB/c.Bcgs mice with peritoneal macrophages from Bcgr C3H/HeN mice. The differential screening of the hybrids was based on the differential sensitivity of Ia expression to suppression with cycloheximide. We found that most of the hybrids expressed Ia without further stimulation with rIFN-gamma. Cycloheximide suppressed the expression of Ia by some of the hybrids. Treatment of these cells with rIFN-gamma resulted in a cycloheximide resistant Ia expression of both parental haplotypes. The macrophage hybrids produced IL-1 beta, IL-1 alpha, and TNF-alpha when stimulated with LPS. There was no correlation between the levels of monokines produced and the persistence of Ia expression. The results of this investigation indicate that the product of the Bcg gene contributed by macrophages from C3H/HeN mice will affect the expression of the I-Ad glycoprotein that is normally transiently expressed by the RAW 309 cell line.     

Correlation between strong adjuvanticity of Klebsiella O3 lipopolysaccharide and its ability to induce lnterleukin-1 secretion

Adjuvant activity of Klebsiella O3 lipopolysaccharide (KO3 LPS) in augmenting antibody response and delayed-type hypersensitivity to protein antigens in SMA mice was much stronger than that of LPS from Escherichia coli O55 and O127 (EO55 LPS and EO127 LPS). Relationship between strength of the adjuvant activity and that of the ability to induce interleukin-1 (IL-1) secretion by peritoneal macrophages from C3H/HeN or SMA mice was investigated using these three kinds of LPS. When supernatant samples of macrophages cultured at 37 °C for 24 hr in the presence of 5 μg/ml LPS were assayed by their mitogenic effect on thymocytes from C3H/HeJ mice, KO3 LPS induced the secretion of about four to six times greater amounts of IL-1 activity than did EO127 LPS. When concentration of LPS used for stimulation of macrophages was varied from 0.1 to 50 μg/ml, KO3 LPS induced the secretion of definitely greater amounts of IL-1 activity than did EO55 LPS and EO127 LPS throughout the LPS concentrations tested. Nearly the same amount of IL-1 activity as that produced by 10 μg/ml EO55 LPS or 50 μg/ml EO127 LPS could be produced by 1.0 μg/ml or lower concentrations of KO3 LPS.     

Granules of human CD3+ large granular lymphocytes contain a macrophage regulating factor(s) that induces macrophage H2O2 production and tumoricidal activity but decreases cell surface Ia antigen expression.

CTL (cytotoxic T lymphocytes) and LGL (large granular lymphocytes) exocytose cytoplasmic granules on activation after recognition of their target, releasing granule-associated molecules. We have previously suggested that this process could release immunoregulatory molecules. In this study we investigated whether normal human LGL granules contained a factor regulating different macrophage activity. Human CD3+ LGL cells were generated by activating peripheral blood lymphocytes (PBL) for 10-12 days with recombinant human IL-2 (rhIL-2), and granules were isolated from disrupted cell homogenate by Percoll gradient fractionation. Solubilized granules were tested for macrophage-activating factor (MAF) activity in three different macrophage assays. When M-CSF-differentiated murine bone marrow-derived macrophages were incubated 9 hr with human LGL granules, they were fully activated to lyse the TNF-resistant P815 tumor cells. The granule-MAF showed a synergistic effect with rhIL-1 beta, rmTNF-alpha, and rmIFN-tau in the cytolytic assay. In addition, proteose-peptone-elicited murine peritoneal macrophages profoundly increased H2O2 production after activation with human LGL granules. However, unlike IFN-tau, no increase in peritoneal macrophage Ia antigen expression was detected after incubation with granules. Moreover, granule-MAF suppressed Ia induction by IFN-tau. These results confirm that human CD3+ LGL granules contain a molecule(s) capable of regulating macrophage function.     

Effects of prostaglandin E2 and nitric oxide inhibitors on the expression of interleukin-10, interleukin-12 and MHC class-II molecules inMycobacterium microti-infected and interferon-γ-treated mouse peritoneal macrophages

Mycobacterium microti-infected mouse peritoneal macrophages produced high amounts of prostaglandin E2 (PGE2) and nitric oxide (NO) when activated with interferon-gamma (IFN-gamma). In order to understand the relation between PGE2 and NO production and the expression of interleukin-12 (IL-12), interleukin-10 (IL-10) and MHC class-II (Ia) molecules by M. microti-infected and IFN-gamma-stimulated macrophages, we analyzed the level of these molecules in the presence or absence of PGE2 and NO inhibitors. Addition of NG-methyl-L-arginine (L-NMA) and indomethacin (IM) caused a significant increase in IL-12 level (2.6- and 1.9-fold, respectively) whereas IL-10 level decreased by 88 and 56%, respectively, relative to M. microti-infected and IFN-gamma-treated control macrophages. Enhanced PGE2 and NO upregulated IL-10 expression and down-regulated IL-12 and MHC class-II (Ia) expression in M. microti-infected and IFN-gamma-treated mouse peritoneal macrophages.     

Combined effects of tumor necrosis factor-alpha, prostaglandin E2, and corticosterone on induced Ia expression on murine macrophages

Ia expression is an important marker of macrophage functional capacity. IFN-gamma induces Ia expression on perhaps all murine macrophages, whereas IL-4, granulocyte-macrophage CSF, and CSF-1 induce Ia on restricted sets of macrophages. Inhibitors of expression include PGE2, glucocorticoids, and IFN-beta. TNF has been found to augment Ia expression on several macrophage lineage cell lines but to inhibit expression on murine peritoneal macrophages. Our study shows that TNF can have opposite effects on Ia expression (induced by IFN-gamma) on thioglycollate-elicited peritoneal macrophages, depending on the length of time cells are treated and on the presence of other modulators. In particular, TNF augmented early expression induced by IFN-gamma but inhibited later expression. And although TNF synergized with PGE2 to markedly inhibit Ia induction on these cells, it partially antagonized the inhibition by corticosterone and IFN-beta. TNF and PGE2 also synergized to inhibit Ia expression induced on bone marrow-derived and splenic macrophages by either IFN-gamma or IL-4. In contrast to their effect on Ia expression, TNF and PGE2 had opposite effects on expression of gamma 2a FcR in macrophages. TNF blocked the increase in FcR expression due to any combination of PGE2, IFN-gamma, and IFN-beta. However, TNF and PGE2 both increased expression of gamma 2a FcR on WEHI-3 cells. If the different effects of TNF reflect the differentiation states of macrophages, its effects on Ia and FcR expression may vary with the progression of an immune response.     

Influence of aging on murine neutrophil and macrophage function against Candida albicans

Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2-3 months old) and aged (18-20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinflammatory cytokines (IL-6, IL-1beta, and tumor necrosis factor-alpha and chemokines (MIP-2) by purified (CD11b(+)) peritoneal macrophages in response to yeasts and hyphae of C. albicans was significantly lower in aged mice as compared with young mice. However, the production of IL-10 by macrophages, in response to C. albicans, was similar in both young and aged animals. Moreover, baseline TLR2 surface expression level was lower on aged macrophages than on control macrophages. Taken together, these data indicate that the increased susceptibility to C. albicans disseminated infections in aged mice is correlated with defects in TLR2 expression and in cytokine production, but not with an impaired fungicidal activity.     

Characterization of interleukin-1 activity in tunicates

1. Eight North American species of tunicates were examined for the presence of interleukin-1 (IL-1) like activity. 2. The tunicates studied produce molecules with readily detectable lymphocyte activation factor (LAF) activity. 3. G50 column chromatography separated molecular species that were directly mitogenic for thymocytes (mol. wt greater than 50,000) from those that were comitogenic in an IL-1 assay (mol. wt 20,000). 4. Tunicate fractions with LAF activity induced increased vascular permeability in rabbit skin. 5. Tunicate LAF activity was neutralized by polyclonal anti-human IL-1 antisera. 6. These data further support the conclusion that IL-1 is an ancient and functionally conserved molecule.     

Cellular origin of blocking factors from cultured spleen cells of tumor-bearing mice

Spleen cells from mice bearing methylcholanthrene-induced tumors were cultured for 2 days without further stimulation. Blocking factors were consistently detected in culture supernatants by their ability to suppress leukocyte adherence inhibition reactions between soluble tumor antigens and peritoneal cells of tumor-bearing mice. The blocking factors were specific for individual tumors. The cellular origin of these factors was investigated by depleting the spleen cell population of various cell types before culturing. The cells involved were removed by treatment with antibodies to certain membrane markers (Thy-1, Ly-2, Ia, I-J) but not by anti-Ly-1 antibodies. Removal of adherent cells also prevented production of blocking factors, which was restored by reconstitution with syngeneic but not allogeneic cells from normal mice. The normal reconstituting cells were shown to bear Ia, but not I-J or IgM. This indicates that blocking factors (previously shown to have I-J determinants in their molecules) originate from suppressor T lymphocytes (Thy-1+, Ly-1-2+, I-J+), with macrophages (I-J-, Ia+) in the role of accessory cells.     

Expression of I-A by macrophages from Bcgr and Bcgs mice. Transient expression of I-A is due to degradation of MHC class II glycoproteins

Murine peritoneal macrophages from mice that are resistant to Mycobacterium bovis (strain BCG) can be induced to continuously express MHC class II (Ia) glycoproteins. In contrast, macrophages from BCG susceptible mice will only transiently express Ia. The purpose of this investigation was to determine the biochemical basis of continuous or transient Ia expression. We therefore compared Ia biosynthesis by macrophages from C.D2Bcgr mice to that by macrophages from congenic BALB/c.Bcgs mice. We show that macrophages from both strains of mice synthesize Ia initially and that very little synthesis occurs after 5 days of in vitro culture. No differences in the amount of Ia synthesized by the macrophages was apparent as determined by quantitative immunoprecipitation and by ELISA. Despite the lack of synthesis, macrophages from C.D2Bcgr mice continue to express Ia. The results of pulse-chase experiments indicate that macrophages from BALB/c.Bcgs mice degrade newly synthesized MHC class II glycoproteins whereas the majority of Ia remains associated with macrophages from the C.D2Bcgr mice. The addition of chloroquine to cultures of macrophages from BALB/c.Bcgs mice prevented the degradation and prolonged the expression of Ia. The results of this investigation show that there are no differences in the synthesis of Ia by macrophages from the two congenic strains of mice. The transient expression of Ia by macrophages from BALB/c.Bcgs mice is due to its degradation.     

Acute infection of mice with lactic dehydrogenase virus (LDV) impairs the antigen-presenting capacity of their macrophages

The infection of mice with lactic dehydrogenase virus (LDV) is characterized by elevated levels of various plasma enzymes such as lactic dehydrogenase, malic dehydrogenase, and others. This elevation is probably the consequence of a defect in the clearance capacity of the virus-affected reticuloendothelial cells, which were found to serve as the targets for LDV infection. Since macrophages play a pivotal role in the induction and regulation of cellular immune responses, we tested the antigen-presenting capacity of macrophages from LDV-infected mice, using a system in which in vitro reactivation of memory T cells depends on specific antigen presentation by macrophages. Our experiments revealed that the antigen-presenting capacity of spleen, lymph node, and peritoneal antigen-presenting macrophages from LDV-infected mice was impaired. This impairment, however, was not due to a defective cellular concentration capacity of antigen, since no difference in the uptake of radiolabeled antigen by uninfected and acutely LDV-infected macrophages was observed. Similarly one cannot attribute the impaired presentation capacity to suppressor cells, since we found that LDV-infected macrophages are not differentially immunosuppressive in the specific in vitro assays used. The analysis of peritoneal macrophages for their expression of Ia antigens revealed that the proportion of Ia-positive macrophages among the LDV-infected peritoneal cells is reduced in comparison to their proportion in noninfected mice. Our results suggest, therefore, that infection of macrophages by LDV is followed by an impairment of their antigen-presenting capacity, probably due to a reduction in the relative proportion of Ia-positive macrophages. These results indicate that the virus either impairs the expression of membrane-associated antigen-presenting components (such as the Ia determinants), thus damaging antigen presentation, or is responsible for the elimination of Ia-positive cells from the peritoneum.     

Structural Characterization of an Alkali-Soluble Polysaccharide from Angelica sinensis and Its Antitumor Activity in Vivo

A novel alkali-soluble polysaccharide (AASP) was isolated from Angelica sinensis (Oliv.) Diels under aqueous alkali treatment, and its structural characterization and antitumor activity in Vivo were evaluated in present study. Results of HPGPC and IC revealed that AASP was a neutral polysaccharide containing Ara, Gal and Glc in the mole ratio of 1.00 : 2.26 : 24.43, with the average molecular weight of 4.7 kDa. Periodate oxidation, Smith degradation, methylation, FT-IR, and NMR analyses further demonstrated that a preliminary structure of AASP was proposed as follows: (1→3)-linked arabinose, (1→6)-linked galactose, and (1→), (1→4), (1→6), (1→3,6)-linked glucose with α- and β-configuration. In Vivo antitumor assays, AASP exhibited prominent antitumor effects on H22 hepatoma cells with an inhibitory ratio of 48.57 % and effectively protected thymuses and spleens of tumor-bearing mice. Besides, AASP displayed a proliferation stimulating activity of immunocytes (splenocytes, peritoneal macrophages and natural killer cells), and an auxo-action for cytokines release (TNF-α, IL-2 and IFN-γ), leading to the apoptosis of H22 solid tumors cells via G0/G1 phase arrested. The above data demonstrated that AASP holds great application potential to be a safe and effective antitumor supplement in the future.     

The immunological mouse mutants nude (nu) and rhino (hr rh ) generate cytotoxic effector cells following adoptive immunotherapy but fail to reject a transplanted tumor

Summary Adoptive immunotherapy, consisting of cyclophosphamide injection and the i. v. transfer of tumor-sensitized T cells, resulted in rejection of the immunogenic fibrosarcoma, MCA/76-9, by syngeneic C57BL/6J (B6) mice. The same treatment of tumor-bearing congenic immunodeficient mice, homozygous for the deleterious mutations nude (nu) and rhino (hr ^rh ), did not result in tumor rejection. Paradoxically, the intratumor and intrasplenic changes taking place in each of the three strains after therapy were indistinguishable. There was an increase in Thy-1⁺, Lyt-2⁺, or L3T4⁺ cells at the tumor site 8 days after adoptive immunotherapy and a similar increase in Thy-1⁺ cells in the spleen. Moreover, the T cells isolated from the tumors or spleens from each genotype were shown to be specifically cytotoxic in vitro as well as in an in vivo Winn assay. Further evidence that immune amplification had occurred in the immunological mutant mice was provided by experiments showing (a) the ability of spleen cells from tumor-bearers and those tested after therapy to produce IL-2 in response to Con A stimulation and (b) an increase in class II-MHC antigen expression by tumor-associated macrophages. The data suggest that, although amplification of antitumor immune responses occurred in the immunological mutants, the absence of a critical host factor limited the potency of the antitumor response. Abbreviations used: CY, cyclophosphamide; TAM, tumor-associated macrophages; TAL, tumor-associated lymphocytes; TAC, tumor-associated cells; B6, C57BL/6J; class II MHC antigens: Ia; con A, concanavalin A; IL-1, IL-2, interleukin 1 and 2