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Molecular basis for the immunosuppressive action of stearic acid on T cells
Abstract
Studies were performed to determine the mechanism by which stearic acid (18:0) selectively inhibits T-dependent immune responses in vitro. Incubation of mitogen-activated B and T cells with 18:0 resulted in dissimilar patterns of incorporation of the saturated fatty acid into their membranes. High-performance liquid chromatography (HPLC) analyses of T cells showed an accumulation of disaturated [corrected] 18:0-containing phosphatidylcholine (PC) that replaced normal cellular PC. Less significant quantities of the same PC species were seen to accumulate in B-cell membranes; rather, they increased their proportion of oleic acid (18:1)-containing PC. The different lipid compositions of the lymphocyte cell membranes after exposure to 18:0 were correlated with their plasma membrane potentials. In T cells, the accumulation of disaturated [corrected], 18:0-containing PC coincided with a rapid (within 8 hr) collapse of membrane integrity, as determined by flow cytometry. The collapse of membrane integrity was found to be time and dose dependent. No such depolarization was observed in B cells which, by virtue of their desaturating ability, were able to avoid incorporating large amounts of disaturated [corrected] 18:0-containing phospholipids into their membranes. It is proposed that a lack of stearoyl-CoA desaturase in T cells precludes them from desaturating exogenously derived 18:0, thus leading to increased proportions of 18:0-containing disaturated [corrected] PC in their cell membranes. The increased abundance of this PC species may enhance membrane rigidity to an extent that plasma membrane integrity is significantly impaired, leading to a loss of membrane potential and ultimately cell function and viability.
... In addition to their roles in helminths' energy flows, lipid metabolites have been recognized as the key players in the regulation of innate immune responses for over 30 years, especially lysophosphatidylglycerols (palmitic and stearic acids) and fatty acyls, such as arachidonic acid (hydroxy-eicosenoic acid) and 2-oxo-docosanoic acid [77]. While some of them mediate the pro-inflammatory cascade initiating PAMPs (pathogen-associated molecular patterns) recognition via TRL4 (Toll-like receptor 4) [78] or serving as a precursor for prostaglandins and leukotrienes [79], such as palmitic and arachidonic acid, respectively, the others (stearic, α-linolenic acid) have immunosuppressive effects on T cells [80]. GINs' strategy mediated through the lipid metabolites and employed for the manipulation of the host immune response towards successful propagation and parasitism, opens a wide perspective that should be compounded by the contemporary use of multi-omics approaches. ...
In nematodes that invade the gastro-intestinal tract of the ruminant, the process of larval exsheathment marks the transition from the free-living to the parasitic stages of these parasites. To investigate the secretome associated with larval exsheathment, a closed in vitro system that effectively reproduces the two basic components of an anaerobic rumen environment (CO2 and 39 °C) was developed to trigger exsheathment in one of the most pathogenic and model gastrointestinal parasitic nematodes, Haemonchus contortus (barber‘s pole worm). This study reports the use of multimodal untargeted metabolomics and lipidomics methodologies to identify the metabolic signatures and compounds secreted during in vitro larval exsheathment in the H. contortus infective third-stage larva (iL3). A combination of statistical and chemoinformatic analyses using three analytical platforms revealed a panel of metabolites detected post exsheathment and associated with amino acids, purines, as well as select organic compounds. The major lipid classes identified by the non-targeted lipidomics method applied were lysophosphatidylglycerols, diglycerides, fatty acyls, glycerophospholipids, and a triglyceride. The identified metabolites may serve as metabolic signatures to improve tractability of parasitic nematodes for characterizing small molecule host–parasite interactions related to pathogenesis, vaccine and drug design, as well as the discovery of metabolic biomarkers.
... The wound healing property was successfully attributed in RM treatment group might be the presence of stearic acid and palmitic acid. The early report showed that the stearic acid immunesuppressive activity on T cell function [54]. Furthermore, palmitic acid has been reported for the regulation of innate immune system [55]. ...
Background: Artemisia absinthium L is an ornamental plant widespread in Saudi Arabia. Traditionally the plant has been used in the Arabic system of medicine. But the scientific evidence of the bioactive compounds and their medicinal value was not yet explored widely.
Objective: The study was designed to analyse the bioactive principles and medicinal properties of Artemisia absinthium L a traditional herb of the southern part of Saudi Arabia.
Method: The bioactive compounds present in hot methanolic extract of the leaves (HMEL) of Artemisia absinthium L. was explored by GC-MS study. The cytotoxicity effect of HMEL was determined against MCF-7 breast cancer cells ATCC and human colon cancer cells HCT 116 ATCC by performing MTT assay. Morphological changes of HMEL treated MCF-7 were observed under a phase-contrast microscope by staining the cells with neutral red. A reaction mixture (RM) of HMEL was prepared in Milli-Q water and antibacterial susceptibility was performed against both Gram-positive and Gram-negative bacteria. Furthermore, In vivo wound healing properties of the RM was screened in male rats and their efficacy was compared with standard povidone iodine cream. Biomarkers such as IL-1β, IL-6, TNF- α, caspase-9 and caspase-3 levels were determined to qualify the wound healing property.
Results: Epiyangambin, flavone, octadecanoic acid, 2,3- dihydroxypropyl ester, palmitic acid β-monoglyceride, á-Dmannofuranoside, camphor, and terpineol were identified as possible compounds through GC-MS analysis. The HMEL of Artemisia absinthium L was actively inhibiting the proliferation of breast cancer cells MCF-7 ATCC at the concentration of 80.96 ± 3.94 µg/ml as IC50 value but failed to inhibit the proliferation against the treated human colon cancer cells HCT 116 cells ATCC. HMEL of Artemisia absinthium L was showing a moderate spectrum of antibacterial effect against the screened bacteria. RM showed better wound healing property than standard povidone iodine cream that modulates cytokine networks and apoptosis markers levels indicated the healing of wound.
Conclusion: The study suggested that novel anticancer, antibacterial and immune modulatory molecules can be developed from the leaves of Artemisia absinthium L.
... There is considerable evidence to show that dietary fat can modulate different immune functions [50][51][52]. These data were supported by several reports that demonstrate speci c in vitro and in vivo effects of saturated and unsaturated fatty acids on lymphocyte function, although the effects of speci c forms of dietary fat on the immune system remain controversial [52][53][54][55]. Furthermore, the enhanced expression of IL-6 was also demonstrated after exposure to long-chain fatty acids in intestinal epithelial cells [56]. ...
Background: Recent studies suggest that endogenous and exogenous free fatty acids play many important roles in mammalian oocyte and preimplantation embryo development. Among these fatty acids, linoleic acid has been reported to affect the apoptosis pathway via nuclear transcription factor-kappa B. The transcription factor NF-κB is a key modulator of apoptosis in a variety of cell types, but to date, this specific function of NF-κB has not been demonstrated in porcine preimplantation embryos. To examine the effect of linoleic acid on parthenogenetic pig embryos produced in vitro, we treated these embryos with linoleic acid at various concentrations to examine the developmental rate, NF‐κB expression, IL-6 expression and apoptosis-related gene mRNA levels.
Results: Linoleic acid had a positive effect on embryo development and was not toxic at a certain concentration, but toxicity was observed at higher concentrations. Furthermore, it was confirmed that the concentration of NF‐κB increased, unlike that of IL-6, as the concentration of linoleic acid increased, and the concentration of NF‐κB was found to increase even at the concentration of linoleic acid at which embryo development decreased. We found that pro-apoptotic gene expression was downregulated in the linoleic acid-treated group. It was also found that BCL-XL, an anti-apoptotic gene, was not affected by linoleic acid, which appears to be an effect of IL-6. In contrast, MCL-1, an anti-apoptotic gene known to be unaffected by IL-6, was found to be increased at the mRNA level in linoleic acid-treated pig embryos. Furthermore, based on both apoptosis and immunocytochemistry staining, as the concentration of NF-kB increased, the nuclear translocation of C-JUN, which is also related to apoptosis, gradually increased, which was dependent on the linoleic acid concentration. It was confirmed that NF-κB is an important factor in the development of porcine embryos by confirming that treatment with a very low concentration of ammonium pyrrolidinedithiocarbamate (APDC, inhibitor of NF-κB) affected NF-κB protein expression, IL-6 protein expression and blastocyst production.
Conclusion: These datas could suggest that porcine embryos can use exogenous linoleic acid as a metabolic energy source via NF-κB. The data also demonstrate the important role of NF-kB in porcine early embryo development.
... SA was reported to selectively inhibit T cell-dependent immune responses in vitro (17,18), and SA accumulation in macrophages can induce Toll-like receptor 4/2-independent inflammation, resulting in endoplasmic reticulum stress-mediated apoptosis (19,20), which could further help prevent aGVHD. However, in animal models, NK cell activity was lower in the spleens from animals fed SA than those fed PA (15). ...
Background:
There are very few studies investigating metabolic biomarkers to predict acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Metabolic models can provide a framework for analyzing the information-rich omics data sets in this setting.
Methods:
Four hundred and fifty-six samples from one hundred and fourteen consecutive patients who underwent HSCT from January 2012 to May 2014 were collected for this study. The changes in serum metabolite levels were investigated using a gas chromatography-mass spectrometry-based metabolomics approach and underwent statistical analysis.
Results:
Significant metabolic changes were observed on day 7. The stearic acid/palmitic acid (SA/PA) ratio was effective in the diagnosis of grade II-IV aGVHD. Multivariate analysis showed that patients with high SA/PA ratios on day 7 after HSCT were less likely to develop II-IV aGVHD than patients with low SA/PA ratios (odds ratio [OR] = 0.06, 95% CI 0.02-0.18, P < 0.001). After the adjustment for clinical characteristics, the SA/PA ratio had no significant effect on overall survival (hazard ratio [HR] = 1.95, 95% CI 0.92-4.14, P = 0.08), and patients in the high SA/PA ratio group were significantly more likely to relapse than those in the low ratio group (HR = 2.26, 95% CI 1.04-4.91, P = 0.04).
Conclusion:
Our findings suggest that the SA/PA ratio on day 7 after HSCT is an excellent biomarker to predict both aGVHD and relapse. The serum SA/PA ratio measured on day 7 after transplantation may improve risk stratification for aGVHD and relapse after allogeneic stem cell transplantation.
Funding:
National Natural Science Foundation of China (81470346, 81773361), Priority Academic Program Development of Jiangsu Higher Education Institutions, Jiangsu Natural Science Foundation (BK20161204), Innovation Capability Development Project of Jiangsu Province (BM2015004), Jiangsu Medical Junior Talent Person award (QNRC2016707), and NIH (AI129582 and NS106170).
... &-Unsaturated fatty acids particularly can disorder the membrane interior and render the cell membrane more rigid (Klausner et al. 1980). T-lymphocytes are particularly susceptible to long-chain fatty acid insertions because, unlike other lymphocytes, they do not possess the 6-desaturase enzyme (Tebbey & Buttke, 1990). The function of certain cell surface receptors is dependent on their lateral mobility within the cell membrane. ...
... 9,[11][12][13][14] Our results in sheep are in agreement with those obtained for other species, which documented negative effects of high concentrations of OA, PA, and SA on lymphocyte functions. [15][16][17][18] Several studies 7,19,20 have been performed in other species to ascertain the mechanisms through which high concentrations of fatty acids impair lymphocyte functions. Addition of OA, PA, and SA to media for culture of lymphocytes induces modifications in cell membrane phospholipids. ...
To assess effects on functions of peripheral blood mononuclear cells (PBMC) obtained from ewes for each of several fatty acids represented in ovine plasma at concentrations mimicking those of ketotic or healthy ewes.
Blood samples obtained from 6 Sardinian ewes.
The PBMC were cultured in media that contained oleic (OA), palmitic (PA), stearic (SA), linoleic (LA), or palmitoleic (POA) acid at concentrations similar to those of ketotic or healthy ewes. Synthesis of DNA was stimulated by use of concanavalin A or pokeweed mitogen (PWM). Secretion of IgM was stimulated by use of PWM.
High concentrations (900, 450, and 225 micromol/L) of OA significantly inhibited DNA synthesis and IgM secretion of PBMC. Conversely, low concentrations (56 or 28 micromol/L) of OA significantly enhanced DNA synthesis of PBMC. High concentrations of PA (600, 300, 150, 75, 375, or 18.7 micromol/L) and SA (300, 150, or 75 micromol/L) significantly inhibited DNA synthesis of PBMC. High concentrations of PA (600, 300, 150, 75, 375, or 18.7 micromol/L) and SA (300, 150, 75, or 38 micromol/L) also significantly inhibited IgM secretion of PBMC. None of the concentrations of LA and POA affected PBMC functions.
Impaired immunoresponsiveness of ketotic ewes is likely associated with an increase of plasma concentrations of OA, PA, or SA and not with that of LA or POA. At physiologic concentrations, single fatty acids are likely to participate in modulation of immunoresponsiveness by exerting suppressive or stimulatory effects on immune cells.
... Similar to SCD1, SCD2 mRNA is expressed to a lesser extent in kidney, spleen, heart and lung where it is induced in response to a high carbohydrate diet [22,34,46]. In addition, SCD2 mRNA is expressed in B-lymphocytrs and is down regulated during T-lymphocyte development474849. In some tissues, such as the adipose and eyelid, both SCD1 and SCD2 genes are expressed whereas in the skin, Harderian and preputial glands all the three gene isoforms are expressed [22,25,27]. ...
Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme catalyzing the synthesis of monounsaturated fatty acids, mainly oleate (18:1) and palmitoleate (16:1). These represent the major monounsaturated fatty acids of membrane phospholipids, triglycerides, wax esters and cholesterol esters. The ratio of saturated to monounsaturated fatty acids affects phospholipid composition and alteration in this ratio has been implicated in a variety of disease states including cardiovascular disease, obesity, diabetes, neurological disease, and cancer. For this reason, the expression of SCD is of physiological significance in both normal and disease states. Several SCD gene isoforms (SCD1, SCD2, SCD3) exist in the mouse and one SCD isoform that is highly homologous to the mouse SCD1 is well characterized in human. The physiological role of each SCD isoform and the reason for having three or more SCD gene isoforms in the rodent genome are currently unknown but could be related the substrate specificities of the isomers and their regulation through tissue-specific expression. The recent studies of asebia mouse strains that have a natural mutation in the SCD1 gene and a mouse model with a targeted disruption of the SCD1 gene have provided clues concerning the role that SCD1 and its endogenous products play in the regulation of metabolism.
We have recently reported that the survival of tilapia, Oreochromis niloticus, during Edwardsiella tarda infection is tightly associated with their metabolome, where the survived O. niloticus has distinct metabolomic profile to dying O. niloticus. Glucose is the key metabolite to distinguish the survival- and dying-metabolome. More importantly, exogenous administration of glucose to the fish greatly enhances their survival for the infection, indicating the functional roles of glucose in metabolome repurposing, known as reprogramming metabolomics. However, the underlying information for the reprogramming is not yet available. Here, GC/MS based metabolomics is used to understand the mechanisms by which how exogenous glucose elevates O. niloticus, anti-infectious ability to E. tarda. Results showed that exogenous glucose promotes stearic acid and palmitic acid biosynthesis but attenuates TCA cycle to potentiate O. niloticus against bacterial infection, which is confirmed by the fact that exogenous stearic acid increases immune protection in O. niloticus against E. tarda infection in a manner of Mx protein. These results indicate that exogenous glucose reprograms O. niloticus anti-infective metabolome that characterizes elevation of stearic acid and palmitic acid and attenuation of the TCA cycle. Therefore, our results proposed a novel mechanism that glucose promotes unsaturated fatty acid biosynthesis to cope with infection, thereby highlighting a potential way of enhancing fish immunity in aquaculture.
While the topics of our NutriNews usually center on issues related to specific nutritional, health or disease conditions, it is appropriate every so often to review and update quality related concerns that surround the dietary supplement industry. This issue will focus on a number of product quality issues and offer independent test results covering topics such as: unsafe lead levels in calcium products, pesticides in Korean ginseng, heavy metal contamination in fish oil, and the presence of magnesium stearate in products. In the absence of independent testing to verify the purity of content, product claims are meaningless. There exists some confusion surrounding the dietary supplement industry, leading to some public misperceptions. The industry is often viewed as having a lack of quality, standards, and account- ability in the manufacturing and sale of dietary supplements. Some feel that the same standards that apply to drugs should apply to dietary supplements, and since those standards are currently different, the quality of dietary supplements should always be in question. At times, the media even portrays the industry as unfocused and unregulated and is critical of many products that reach the store shelves. While it is true that dietary supplements are not drugs or over- the-counter medications, they are regulated, although differently from the way the Food and Drug Administration (FDA) regulates pharmaceuticals. Without getting into a cumbersome review of reg- ulatory law, it is important to understand a number of key concepts.
Apoptosis and underlying mechanisms were evaluated in human umbilical vein endothelial cells (HUVECs), in target tissues of late diabetic vascular complications [human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs)], and in endothelial progenitor cells (EPCs) exposed to FFAs, which are elevated in obesity and diabetes. Saturated stearic acid concentration dependently induced apoptosis that could be mediated via reduced membrane fluidity, because both apoptosis and membrane rigidity are counteracted by eicosapentaenoic acid. PUFAs triggered apoptosis at a concentration of 300 micromol/l in HUVECs, HAECs, and EPCs, but not HRECs, and, in contrast to stearic acid, involved caspase-8 activation. PUFA-induced apoptosis, but not stearic acid-induced apoptosis, strictly correlated (P < 0.01) with protein expression of E2F-1 (r = 0.878) and c-myc (r = 0.966). Lack of c-myc expression and activity owing to quiescence or transfection with dominant negative In373-Myc, respectively, renders HUVECs resistant to PUFA-induced apoptosis. Because c-myc is abundant in growing cells only, apoptosis triggered by PUFAs, but not by saturated stearic acid, obviously depends on the growth/proliferation status of the cells. Finally, this study shows that FFA-induced apoptosis depends on the vascular origin and growth/proliferation status of endothelial cells, and that saturated stearic acid-induced apoptosis and PUFA-induced apoptosis are mediated via different mechanisms.
Studies were conducted to determine the effects of varying in vitro temperatures during cellular processing of T-dependent antigens by catfish antigen-presenting cells (APC). Strategy involved pulsing of long-term monocyte lines (used as APC) with antigen at different temperatures for various periods of time and then fixing and coculturing with autologous peripheral blood leukocytes (PBL) as responders at a permissive temperature (i.e., 27 degrees C). Results showed that APC incubated with antigen at low, nonpermissive, but physiologically relevant, temperatures (11 and 17 degrees C) elicited secondary proliferative responses by autologous PBL. However, responses elicited with APC pulsed at 11 and 17 degrees C required longer exposure to the antigen prior to fixation (i.e., greater than or equal to 8 h compared to 5 h, the optimal incubation time at 27 degrees C). Further, there was sufficient cell-associated antigen during a short-term pulsing period (1-2 h) at 11 and 17 degrees C to provide efficient presentation after subsequent incubation of the APC at 27 degrees C for an additional 10 h before fixation. In contrast, APC pulsed with antigen at an extremely low, physiologically irrelevant, temperature (4 degrees C) for extended periods of time did not elicit the proliferation of autologous responders unless antigen pulsing was carried out for 1-2 h and the APC subsequently shifted to 27 degrees C for an additional 10 hr. Antigen uptake assays revealed significant and similar levels of internalized antigen at 11, 17, and 27 degrees C, but not at 4 degrees C. However, intracellular degradation and formation of trichloroacetic acid-soluble antigen catabolites at 11 and 17 degrees C appeared to occur at a slower rate compared to APC at 27 degrees C. Significantly, primary anti-hapten plaque-forming cell responses were also observed with PBL cocultured with APC pulsed with hapten-carrier conjugates at 11, 17, and 27 degrees C. Consequently, the previously observed suppression of primary T-cell responses in fish at low, physiologically relevant, temperatures is not due to impaired antigen processing or presentation.
B lymphocytes from the spleens of normal (BALB/c) and autoimmune (MRL/lpr) strains of mice express the SCD-2 form of stearoyl-CoA desaturase as opposed to the SCD-1 form of the gene which is expressed in liver. However, whereas BALB/c T cells did not express SCD-1 or SCD-2, both BALB/c thymocytes and MRL/lpr T cells expressed SCD-2, suggesting a developmental down-regulation of SCD-2 within the T cell lineage. Northern analyses also revealed the expression of SCD-2 in the T cell lines BW5147, CTLL-2 and HT-2 and in BCL1, a B cell line. SCD-1 expression was not detected in any of the lymphoid cells tested. Finally, we show that SCD-2 gene expression is inhibited by arachidonic acid (20:4). These results demonstrate the complexity of SCD-2 regulation in lymphoid cells.
This work was based upon the observation that a reduction in the level of serum, provided to murine lymphocytes in culture, augmented endogenous unsaturated fatty acid (UFA) synthesis. Since the phospholipids of BW5147 cells grown in 1% serum were especially deficient in arachidonic acid (20:4), and given the findings of previous workers, we suspected that the availability of exogenous 20:4 in serum might correlate with the squelching of UFA synthesis. Indeed, after a 5 h exposure to 4-28 microM 20:4, the 20:4 content of BW5147 cell phospholipids increased from 1% to 15% of the total fatty acids with a coincident reduction in 18:1 synthesis to approx. 30% of starting values. Subsequent studies were done to define the mechanism by which 20:4 down-regulates 18:1 synthesis. The results indicated that 20:4 inhibited endogenous 18:1 synthesis by reducing stearoyl-CoA desaturase (SCD) enzyme activity. Moreover, as determined by Northern blot analyses, the inhibitory effect of 20:4 on stearoyl-CoA desaturase activity coincided with decreased stearoyl-CoA desaturase mRNA levels. Exposure of BW5147 cells to either 20:4, actinomycin D, or both, resulted in a temporal decay of stearoyl-CoA desaturase mRNAs with half-lives ranging from 4.0 h to 4.4 h. Such a similarity in decay times implied that 20:4 regulates stearoyl-CoA desaturase expression by inhibiting transcription. This was confirmed by nuclear run-on studies in which 20:4 was found to inhibit transcription of nascent stearoyl-CoA desaturase mRNA. Collectively, these findings implicate 20:4 as an important regulator of stearoyl-CoA desaturase gene expression, and hence UFA synthesis, in lymphoid cells.
A prospective randomized study of the immunological effects of three total parenteral nutrition (TPN) regimens in patients undergoing preoperative parenteral nutrition was conducted. In one regimen the calories were derived solely from glucose. The others were identical except that 50 per cent of the calories were provided as lipid emulsion, in one as long-chain triglycerides (LCT) only while the other contained half the fat as medium-chain triglycerides (MCT) and half as LCT (MCT/LCT). Natural killer (NK) activity and lymphokine-activated killer (LAK) activity were significantly higher after TPN with the MCT/LCT solution. A significant fall in LAK activity occurred after TPN with the LCT solution. The interleukin 2 content in supernatants from activated T lymphocytes was significantly higher after TPN with the LCT-containing solution. Solutions containing LCT and those containing MCT perturb cytokine interactions, but this is less with MCT-containing solutions, which may augment certain responses. These observations may have implications for the design of TPN regimens.
1. Studies were performed to determine if the inability of murine T cells to provide primary helper function at low temperature (27 degrees C) could be correlated with their inability to synthesize unsaturated fatty acids (UFAs). 2. In the absence of exogenous oleic acid (18:1), splenocytes responded to a T-dependent (TD) Ag (trinitrophenyl-keyhole limpet hemocyanin, TNP-KLH) at 37 degrees C but not at 27 degrees C. The addition of 150 microM 18:1 almost completely restored plaque-forming cell (PFC) responses to TNP-KLH at 27 degrees C but markedly suppressed PFC responses to the TD Ag at 37 degrees C. 3. During incubation at 27 degrees C, B cells converted 3- to 5-fold more stearic acid (18:0) to 18:1 and showed a greater accumulation of monounsaturated phospholipid molecular species than did T cells. 4. Following incubation in the presence of a rescuing dose of 18:1 (150 microM), both B and T cells accumulated large amounts of dioleoyl PC. 5. It is proposed that the absence of 18:1 synthesis in T cells is responsible for the unique low temperature susceptibility of this lymphocyte population.
We have previously shown that albumin-complexed stearic acid (18:0) inhibited in vitro primary anti-TNP plaque-forming cell (PFC) responses to trinitrophenyl keyhole limpet hemocyanin (TNP-KLH), but did not affect primary PFC responses to trinitrophenyl lipopolysaccharide (TNP-LPS). The present studies were done to identify the cellular target of fatty acid inhibition. The addition of 18:0 at the initiation of antibody cultures exerted a dose-dependent inhibitory effect on subsequent PFC responses to TNP-KLH, and removal of the fatty acid after 20 h did not reverse its inhibitory effect. Preincubation of isolated T-cells with TNP-KLH and 18:0 resulted in a similar inhibition of subsequent PFC responses, but a preincubation of isolated B-cells had no effect. The addition of 18:0 to the culture system in vitro led to a marked reduction in the level of IL-2 detectable in culture supernatants, and PFC responses could be restored by providing exogenous mouse recombinant IL-2. The addition of antigen-primed T-helper cells to antibody cultures partially abrogated the inhibition by 150 microM 18:0, apparently due to their greater production of IL-2. Lastly, following overnight incubation of unfractionated splenic lymphocytes in the presence of TNP-KLH and [1-14C]-18:0, B-cells were shown to contain nearly 5-fold more radiolabeled oleic acid (18:1) than T-cells. Collectively, these findings implicate T-helper cells as the principle target of 18:0-inhibition of primary antibody responses in vitro, possibly as a result of the inability of T-helper cells to avoid an over accumulation of stearic acid in their membrane phospholipids.
Effects of palmitic, stearic, oleic, and linoleic acid on mitogen-induced DNA synthesis, on production of IL-1 beta, IL-2, IFN-gamma, and TNF-alpha, and on IL-2R expression were determined in human peripheral lymphocytes. Free fatty acids (FFA) were added over a wide range of concentrations to cells cultured under serum free conditions with fatty acid free albumin. DNA synthesis was stimulated by low and inhibited by high FFA concentrations. Physiological concentrations were stimulatory, except for linoleic acid. Cytokine production became affected by all FFA tested. Palmitic acid enhanced the release of IFN-gamma at concentrations that diminished TNF-alpha production. Saturated fatty acids were significantly more potent than unsaturated fatty acids in affecting cytokine production. IFN-gamma secretion was significantly more stimulated or inhibited by the various FFA compared with the other cytokines. IL-2R expression correlated with the production of IL-2. When tested in combination, stimulatory as well as inhibitory effects of the individual FFA became attenuated. It is suggested that palmitic, stearic, oleic, and linoleic acid are physiological regulators of DNA synthesis and cytokine release in human peripheral lymphocytes. Modulation of FFA ratios may be an effective means for the fine tuning of the immune system. As secretory mechanisms of cytokines appear to exhibit substrate specificity for FFA, the release of individual cytokines may be selectively influenced by FFA.
The effect of long chain and medium chain fatty acid absorption on transport and mitogen-induced blast transformation of lymphocytes in intestinal lymphatics was investigated. Intestinal lymph was collected from mesenteric lymph duct cannulated rats maintained in Bollman's cage. Following the intraduodenal administration of oleic acid (long chain fatty acid) or octanoic acid (medium chain fatty acid), only oleic acid produced a significant increase in lymphocyte flux and enhanced proliferative response of lymphocyte in intestinal lymph, without significant alteration of lymphocyte subsets. These changes appeared to be closely correlated with the appearance of radiolabelled oleic acid. Absorption of a medium chain fatty acid, octanoic acid, most of which appeared to be transported to portal blood, did not produce a significant elevation of lymphocyte flux or increased proliferative response of lymphocyte in intestinal lymph. Pluronic L-81, which is a potent inhibitor of the intracellular formation and secretion of chylomicron, significantly attenuated the increased lymphocyte flux and suppressed the enhancement of lymphocyte responsiveness to PHA in intestinal lymph after stimulation by oleic acid administration. There is a possibility that lymphocyte transport and proliferative response in intestinal lymph during oleic acid absorption are closely related to the process of chylomicron formation and secretion to lymphatics in the intestinal mucosa.
The functional relationships between lymphoid cells and the adipose tissue that surrounds lymph nodes were investigated in healthy adult guinea pigs. Lymphoid cells extracted from healthy adult guinea pigs were co-cultured for 48 h with adipose tissue explants from 18 sites defined by their anatomical relations to lymph nodes. Such explants from near a node suppressed lymphocyte proliferation stimulated with concanavalin A or lipopolysaccharide more than those from sites 5-10 mm from nodes. Inhibition was almost completely abolished by 500 microU insulin. The presence of lymphoid cells increased lipolysis (measured as glycerol release) in adipose tissue from all depots containing lymph nodes (i.e., except perirenal), especially in the presence of mitogens and with near-node samples from intermuscular and mesenteric depots. Inhibition of lymphocyte proliferation by adipose tissue was proportional to the additional lipolysis stimulated by the presence of lymphoid cells. For all depots except the mesenteric, glycerol release stimulated by lymphoid cells was inversely proportional to spontaneous lipolysis in adipose tissue cultured alone. These experiments demonstrate reciprocal interactions between lymphoid cells and adipose tissue, especially that around lymph nodes. The mediators of the action of adipose tissue on lymphoid cells probably include lipolytic products; mediators of the inverse effects are unknown.
A novel approach for immunocontraception by intervention of local cell mediated immunity in the reproductive system by using single intrauterine application of neem oil has been described earlier. The reversible block in fertility was reported to last for 107-180 days in female Wistar rats and 7 - 11 months in monkeys. The present study, describes the identification and characterization of the biologically active fraction from neem seeds (Aradirachta indica A. Juss. Family Meliaceae), responsible for the above activity in adult female Wistar rats. Initial studies with the mechanically extracted oil and solvent extracts of neem seeds have revealed that the antifertility activity was present in constituents of low to intermediate polarity. A hexane extract of neem seeds was reported to be biologically active. Subsequently, hexane extract was sequentially fractionated through the last active fraction using various separation techniques and tested for antifertility activity at each step. Preparative HPLC was used for isolating individual components of the active fraction in quantities, sufficient for characterization. An analytical HPLC method was developed for standardization of the fraction. The active fraction was identified to be a mixture of six components, which comprises of saturated, mono and di-unsaturated free fatty acids and their methyl esters. Dose response study was performed with the last active fractions. The antifertility activity with the active fraction was reversible in nature and it was completely active until 5% concentration. There was no systemic toxic effect following the administration of the active fraction. This study, for the first time, proposes an active fraction from neem seeds, responsible for long term and reversible blocking of fertility after a single intrauterine administration with high efficacy.
The purpose of this randomized study was to compare the efficacy of medium chain triglycerides (MCT) plus long chain triglycerides (LCT) with LCT alone in total parenteral nutrition (TPN) solutions in patients with various hematologic malignancies who underwent a hematopoietic peripheral blood stem cell (PBSC) transplantation.
Of 36 patients entering into this study, 18 received MCT + LCT (group I) and the remaining 18 received LCT alone (group II) in TPN solutions. Patients were comparable regarding age, gender, donor-recipient gender, diagnosis, body weights, blood group differences and number of infused CD34(+) cells/kg. Post - transplant parameters such as duration of platelet and neutrophil engraftment, coagulation parameters, number of days of febrile neutropenia and antibiotic administration, plasma glucose, triglyceride, cholesterol and albumin levels, graft-versus-host disease (GVHD) and first 100 day mortality were compared in both groups.
Median days of neutrophil >0.5 x 10(9)/l and platelet of >20 x 10(9)/l in group I and group II were 15 (range, 8-21), 11 (10-29) and 14 (range, 9-31), 13 (9-18) respectively (P>0.05). Median days of febrile neutropenia in group I and II were 10 (range, 4-23) and 7 (2-13) respectively (P=0.01). Median days of antibiotic administration in group I and II were 12 (range, 6-22) and 8 (4-25) respectively (P=0.04). Pre, peri- and post-transplant coagulation parameters such as PT, aPTT, and fibrinogen did not differ significantly between two groups (P>0.05), as well as plasma glucose, triglyceride, cholesterol, albumin levels, GVHD and first 100 day mortality.
There was no difference between patients receiving MCT + LCT (group I) and LCT alone (group II) in TPN solutions regarding duration of engraftment and coagulation parameters, but numbers of median days of febrile neutropenia and days of antibiotic administration were significantly shorter in patients receiving LCT alone (group II) than those receiving MCT + LCT (P<0.01 and 0.04 respectively).
Intestinal mucosal immunity is modulated by cytokine release from intestinal cells, but little is known about the relation between nutrient absorption and cytokine release. In this study, we examined how exposure to fatty acids affects the production of growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) and interleukin (IL)-6 in rat intestinal epithelial cells (IEC). The long-chain fatty acids, oleic, linoleic and arachidonic acids, and the middle-chain fatty acid octanoic acid were administered to subconfluent cultures of IEC-6 cells alone, or in combination with IL-1beta and transforming growth factor (TGF)-beta. The GRO/CINC-1 and IL-6 concentrations in culture media were determined by sandwich enzyme immunoassay. In epithelial cells, GRO/CINC-1 and IL-6 mRNA expression were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and mitogen-activated protein kinase (MAPK) activities determined by immunoblotting. Administration of long-chain fatty acids significantly increased the GRO/CINC-1 and IL-6 secretion into culture media, and this secretion was markedly increased (P < 0.05) in the presence of IL-1beta or TGF-beta. Octanoic acid had no effect on GRO/CINC-1 or IL-6 production. Furthermore, treatment with long-chain fatty acids significantly enhanced the GRO/CINC-1 and IL-6 expression that was induced by IL-1beta or TGF-beta. MAPK activity was significantly enhanced by treatment with long-chain fatty acids. Inhibitors of phospholipase C, protein kinase C or MAPK significantly reduced the fatty acid-induced increase in GRO/CINC-1 secretion, whereas a calcium/calmodulin inhibitor did not attenuate the secretion. These results suggest that long-chain fatty acids enhance cytokine release under conditions of inflammatory stimulation in the intestinal mucosa.
Intraepithelial lymphocytes (IELs) play important roles in intestinal mucosal immunity. Although fatty acids are known to modulate the functions of immune effector cells, there has been no information about how fat exposure affects immunological function of IELs. In this study, we examined how fatty acids of various chain lengths modulate the production of interferon (IFN)-gamma by IELs stimulated with T-cell receptor (TCR) or interleukin (IL)-12/IL-18. IELs isolated from the small intestine of BALB/c mice were stimulated with plate-coated anti-CD3 monoclonal antibody (mAb) or IL-12/IL-18. They were coincubated in microtiter plates for 3 days with various concentrations of fatty acid micelles. We used arachidonic acid, linoleic acid, and oleic acid as long-chain fatty acids, and used octanoic acid as a medium-chain fatty acid. IFN-gamma in the supernatants were measured by ELISA, and the expression of IFN-gamma mRNA in IELs was determined by RT-PCR. Significant production of IFN-gamma from IELs was observed after anti-CD3 mAb stimulation. The combination of IL-12 and IL-18 induced significant levels of IFN-gamma production without TCR stimulation. Increased IFN-gamma mRNA was also observed after anti-CD3 or IL-12/IL-18 stimulation. Long-chain fatty acids dose-dependently inhibited the stimulated-IFN-gamma production at concentrations greater than 10 micro M, but the medium-chain fatty acid did not cause any significant changes in IFN-gamma production. IFN-gamma production from gammadelta IELs was very low compared with alphabeta IELs, however, both populations showed similar attenuating patterns when treated with long-chain fatty acids. There is a possibility that the exposure of IELs to intraluminal fatty acids significantly modifies the immune function of intestinal mucosa.
Detailed analyses regarding the effects of temperature and phospholipid fatty acid replacement on the capping of membrane immunoglobulin (mIg) have been performed using a recently described flow cytometric procedure (Cuchens, M. A., and Buttke, T. M. (1984) Cytometry 5, 601-609). Purified murine B cells were incubated for 12-20 h in the presence of bovine serum albumin-complexed 80 microM stearic (18:0), oleic (cis-18:1), or linoleic (cis, cis-18:2) free fatty acids. Unmodified and free fatty acid-treated cells were stained with fluorescein-conjugated rabbit anti-mouse Ig and subjected to pulse-shape (width) analyses to follow the kinetics of mIg capping. In both unmodified and free fatty acid-treated cells, capping of mIg occurred at all temperatures between 17 and 37 degrees C, but the rate of cap formation was temperature dependent. Arrhenius plots of mIg capping were linear, with activation energies ranging from 14 to 23 kcal/mol depending on the saturated/unsaturated fatty acid ratio of B cell phospholipids. Ligand-induced redistribution of mIg thus appears to be sensitive to changes in membrane acyl chain composition.
The response of several Escherichia coli membranous enzymes to hydrolysis of up to 95% of membrane phospholipid has been investigated. Purified phospholipase C of Bacillus cereus was utilized in these studies. The rate and extent of digestion of E. coli phospholipids was independent of whether the lipid was associated with membrane protein or extracted from membranes and sonically dispersed. Phosphatidylethanolamine and phosphatidylglycerol were completely hydrolyzed, while cardiolipin was partially resistant to hydrolysis by phospholipase C. Acyl-CoA:glycerol 3-phosphate acyltransferase and NADH oxidase were inactivated at a rate very similar to the rate of hydrolysis of total lipids. Acyl-CoA:1-acylglycerol 3-phosphate acyltransferase was inactivated to an extent of 50% during hydrolysis of 50% of membrane phospholipid. The remaining activity was stable to continued hydrolysis of phospholipid. Glycerol 3-phosphate dehydrogenase and succinic dehydrogenase remained completely active after hydrolysis of 95% of membrane phospholipids. These results show the heterogeneity of membranous enzymes with respect to their dependence upon the presence of intact membrane phospholipids. The lack of effect of membranous lipid-protein interactions on the accessibility of phospholipids to phospholipase C hydrolysis was shown in general by the similarity in the rates of hydrolysis by phospholipase C of membranous and isolated phospholipids. More specifically, the similarity in the rate of hydrolysis of phospholipids and the rates of inactivation of certain membranous enzymes suggests that these enzymes are dependent on phospholipids whose susceptibility to phospholipase C hydrolysis is similar to the bulk of membrane phospholipids.
Cyclosporin A (CsA) is a potent immunosuppressive agent, now gaining wide application in human organ transplantation. The immunosuppressive activity of CsA is at least in part due to inhibition of lymphokine production by activated T lymphocytes. Specifically, inhibition of T-cell growth factor (TCGF; also designated interleukin 2) production appears to be an important pathway by which CsA impairs T-cell function. To define further both the specificity of CsA and the level at which it interferes with lymphokine gene expression, we have studied its effects on TCGF mRNA accumulation as well as TCGF gene transcription. These studies were performed with a cloned human leukemic T-cell line (Jurkat, subclone 32), which can be induced with phytohemagglutinin and phorbol 12-myristate 13-acetate to produce large amounts of TCGF. In these cells, high levels of TCGF mRNA were present in induced but not in uninduced Jurkat cells as judged by hybridization to a cloned human TCGF cDNA probe. CsA completely inhibited induced TCGF mRNA accumulation at concentrations of 0.3-1.0 microgram/ml, whereas low levels of appropriately sized TCGF mRNA were present at 0.01 microgram/ml. In nuclear transcription experiments, CsA inhibited the synthesis of TCGF transcripts in a dose-dependent manner with complete inhibition at a concentration of 1 microgram/ml. In contrast, CsA did not inhibit the expression of two other inducible genes, TCGF receptor and HT-3. Further, HLA gene expression was also less affected than TCGF in CsA-treated cells. These data suggest a relatively selective action of CsA on TCGF gene transcription.
Cyclosporine, a cyclic endecapeptide of fungal origin, has recently been released for use in clinical transplantation. Trials in kidney, heart, liver and bone marrow recipients were encouraging: 1-year graft survival rates were 70% to 80% for kidney and heart recipients, and 60% to 65% for liver allograft recipients. Cyclosporine is also effective in treating bone marrow recipients with acute graft-versus-host disease. The drug selectively inhibits T-helper cell production of growth factors essential for B cell and cytotoxic T-cell differentiation and proliferation, while allowing expansion of suppressor T-cell populations. Drug absorption varies greatly, necessitating monitoring of drug level and individualization of therapy. Nephrotoxicity is the most frequent side effect of cyclosporine. An increased incidence of B-cell lymphomas seen when cyclosporine was used in conjunction with cytotoxic agents or anti-lymphocyte globulin has very rarely been observed when concomitant immunosuppression has been limited to low-dose corticosteroids. Lower initial doses of cyclosporine, followed by more rapid tapering may reduce the incidence of nephrotoxicity without compromising improved graft outcome.
Certain polyunsaturated fatty acids cannot be synthesized by mammals and are therefore important factors in the mammalian diet. As components of the phospholipids of the plasma membrane, and as precursors of prostaglandins they are essential for the growth and function of all tissues. The major essential fatty acid (EFA) is linoleic acid, which serves as the parent substance for the ω-6 family of EFA. Other members of this family — in the sequence of their biosynthesis — are γ-linolenic, dihomo-γ-linolenic, and arachidonic acid.
Cyclosporin A (CsA) produced dose-dependent membrane depolarization of human peripheral blood lymphocytes. The phenomenon was investigated applying the membrane potential probe dihexyloxacarbocyanine iodide in a flow cytometer in combination with ionophores, hormones and monoclonal antibodies binding to different subclasses of lymphocytes and the anti-interleukin 2 receptor antibody. Human interferon- abolished the depolarizing effect of cyclosporin on lymphocytes. Interleukin 2 caused depolarization and also enhanced the effect of CsA. OKT4 and OKT8 monoclonal antibodies slightly hindered depolarization by CsA while OKT3, OKT11 and OKIa1 antibodies had no such effect. Valinomycin decreased CsA's effect on the membrane potential while the ionophore A-23187 and ionomycin caused depolarizations that were additive with CsA's. CsA treatment released the isotope from 42K-loaded human lymphocytes in a dose-dependent fashion. CsA addition increased intracellular calcium content. CsA decreased the motional freedom of a spin probe in the membrane, but did not hinder the binding of fluoresceinated antibodies to the cell surface. These results suggest immediate alteration in membrane structure upon CsA treatment, causing potassium leakage and calcium ion uptake. These are the earliest detected effects of CsA on cells so far.
The fungus metabolite cyclosporin A is a small peptide acting as a novel antilymphocytic agent. It strongly depressed appearance of both direct and indirect plaque-forming cells and produced a clear dose-dependent inhibition of haemagglutinin formation in mice upon oral administration. Skin graft rejection in mice and graft-versus-host disease in mice and rats were considerably delayed by cycloporin A which also prevented the occurrence of paralysis in rats with experimental allergic encephalomyelitis. This compound was not only highly effective in preventing development of Freund's adjuvant arthritis, but in addition improved the symptoms in rats with established arthritis, although it is inactive in acute inflammation. This new agent contrasts with other immunosuppressives and cytostatic drugs in its weak myelotoxicity. Experimental evidence suggests that cyclosporin A, rather than being cytostatic or lympholytic, affects an early stage of mitogenic triggering of the immunocompetent lymphoid cell.
The membrane potentials of individual cells can be estimated by flow cytometric quantitation of the cells' uptake of the fluorescent lipophilic cationic dye 3,3'-dihexyloxacarbocyanine iodide. Human lymphocytes separated from peripheral blood on Hypaque-Ficoll gradients are uniformly depolarized by gramicidin and hyperpolarized by valinomycin. Concanavalin A and phytohemagglutinin depolarize only a fraction of the lymphocytes. The flow cytometric technique allows precise detection of heterogeneous membrane potential responses to stimuli such as lectins; it could also provide a basis for sorting cells that respond differently to a given stimulus.
Mouse peritoneal macrophages incubated in serumless medium containing a 19:0 or trans-18:1 fatty acid complexed to bovine serum albumin incorporate the exogenous fatty acid supplement into cellular phospholipids. Within 8 hr, 25% of the total phospholipid fatty acids are derived from the supplement, with cell viability remaining greater than 95%. The incorporation of either of these supplements increases the saturated/unsaturated fatty acid ratio in the phospholipids 2-fold over that of cells cultured in serum and effects striking changes in endocytic activities. The levels of both fluid-phase pinocytosis and receptor-mediated phagocytosis are decreased at all temperatures examined between 15 degrees and 37 degrees. The increased degree of saturation of cell phospholipids correlates with decreased endocytic rates for both processes and with increased activation energies (Eact) for phagocytosis. The Eact values for phagocytosis, which range from 54 to 90 kcal/mol, depend on the supplementation conditions used. Although the levels of pinocytosis are depressed, the Eact values for pinocytosis (17--25 kcal/mol) are not strikingly affected by saturated fatty acid enrichment. These observations suggest that the degree of lipid fluidity of macrophage membranes influences both phagocytosis and pinocytosis in macrophages.
This chapter discusses fatty acids and immunity. Like all mammalian cells, lymphocytes contain a variety of fatty acids, some of which, the essential fatty acids, must be supplied to the cells because they are unable to synthesize them. Of the essential fatty acids, linoleic acid and the derivative arachidonic acid are major components of the phospholipids of lymphocyte membranes, while α-linolenic and its derivatives are only minor components. A wide variety of fatty acids produce effects on both the lymphoid and reticuloendothelial systems; the actual effects produced depend on the method of administration and the chemical nature of the fatty acid. Arachidonic acid is an immediate prostaglandin precursor and plays a central role in lymphocyte activation. Membrane phospholipid fatty-acid composition alters when lymphocytes are stimulated, either by antigen or by mitogens, such as phytohaemagglutinin or concanavalin A. Increased turnover of phospholipid fatty acids, following lymphocyte stimulation, requires a transferase to incorporate new fatty acids and a means of generating lysophosphatides and free fatty acids as substrates for this enzyme.
We have previously shown that albumin-complexed stearic acid (18:0) inhibited in vitro primary anti-TNP plaque-forming cell (PFC) responses to trinitrophenyl keyhole limpet hemocyanin (TNP-KLH), but did not affect primary PFC responses to trinitrophenyl lipopolysaccharide (TNP-LPS). The present studies were done to identify the cellular target of fatty acid inhibition. The addition of 18:0 at the initiation of antibody cultures exerted a dose-dependent inhibitory effect on subsequent PFC responses to TNP-KLH, and removal of the fatty acid after 20 h did not reverse its inhibitory effect. Preincubation of isolated T-cells with TNP-KLH and 18:0 resulted in a similar inhibition of subsequent PFC responses, but a preincubation of isolated B-cells had no effect. The addition of 18:0 to the culture system in vitro led to a marked reduction in the level of IL-2 detectable in culture supernatants, and PFC responses could be restored by providing exogenous mouse recombinant IL-2. The addition of antigen-primed T-helper cells to antibody cultures partially abrogated the inhibition by 150 microM 18:0, apparently due to their greater production of IL-2. Lastly, following overnight incubation of unfractionated splenic lymphocytes in the presence of TNP-KLH and [1-14C]-18:0, B-cells were shown to contain nearly 5-fold more radiolabeled oleic acid (18:1) than T-cells. Collectively, these findings implicate T-helper cells as the principle target of 18:0-inhibition of primary antibody responses in vitro, possibly as a result of the inability of T-helper cells to avoid an over accumulation of stearic acid in their membrane phospholipids.
Stearic acid is toxic for T lymphocytes in vitro but has little effect on B lymphocytes. To investigate the molecular basis for this difference, purified murine T and B lymphocytes were compared for their abilities to incorporate and metabolize stearic acid. Unstimulated T and B cells incorporated identical amounts of stearic acid into six different phospholipids and four neutral lipids. After mitogen stimulation, fatty acid uptake was increased in both lymphocyte types, but cell-specific differences were seen in the distribution of stearic acid among the various cellular lipids. Doses of stearic acid that selectively inhibited T-cell proliferation resulted in a 5-fold greater accumulation of distearoylphosphatidylcholine in T cells than in B cells. Whereas T cells did not desaturate the exogenously derived stearic acid, up to 25% of the saturated fatty acid was converted to oleic acid in B cells. These findings suggested a deficiency of stearoyl-CoA desaturase (acyl-CoA, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.5) activity in T cells, which was confirmed by subsequent studies. Cell-free extracts from B cells displayed nearly 20-fold more stearoyl-CoA desaturase activity than T-cell extracts, and the level of stearoyl-CoA desaturase mRNA was 30-fold higher in B cells. Collectively, our data indicate that murine T cells are deficient in unsaturated fatty acid synthesis. The deficiency of stearoyl-CoA desaturase in T cells may represent the basis for the differing sensitivities of T and B lymphocytes to inhibition by saturated fatty acids.
Free fatty acids (FFA) were tested for their effects on in vitro thymus-dependent (TD) and thymus-independent (TI) immune responses. Murine T cells proliferating in one-way mixed leucocyte reactions (MLR) were extremely sensitive to inhibition by exogenous stearic acid (18:0) but were only moderately affected by oleic acid (18:1). T-cell proliferation was suppressed when 18:0 was added as late as 44 hr after allogeneic stimulation, but sensitivity to 18:1 was limited to the first 30 hr of culture. The inhibitory effects of 18:0, but not 18:1 were potentiated by concomitant T-cell activation and under such conditions the effects of 18:0 were irreversible within 5 hr. The two fatty acids were additionally tested for their effects on the anti-hapten antibody-secreting cell responses to TD and TI antigens. Both 18:0 and 18:1 inhibited the primary antibody response elicited by a TD antigen (TNP-KLH) but neither fatty acid significantly affected the primary antibody response to a TI antigen (TNP-LPS). Following in vivo immunization with TNP-KLH, isolated spleen cells were challenged with the same antigen in vitro in the presence of FFA. Whereas 18:1 had little effect on the secondary immune response, the addition of 18:0 led to a 3-4-fold increase in the number of anti-TNP plaque forming cells. Further studies showed that TNP-KLH-induced T cell proliferation was potently inhibited by 18:0 but 18:1 had no effect. These results suggest that an inhibition of T-cell proliferation is the primary way in which 18:0 modulates TD immune responses in vitro. By contrast, 18:1 appears to inhibit primary antibody responses and MLR via alternative mechanisms.
Cyclosporin (CsA) therapy has improved the outcome of allotransplants. Because of a relatively selective action on T lymphocytes, CsA therapy causes fewer immunosuppressive complications of infection or malignancy compared to previous chemical agents, which were relatively nonspecific in their spectrum of action on lymphoid versus nonlymphoid cells. Gastrointestinal complaints after oral administration and vasomotor reactions after intravenous administration represent pharmacologic toxicities rarely of major clinical import. Drug-induced complications involve primarily the neuroectodermal, or the mesenchymal hepatic and renal, systems. While the former group of complications are rarely severe, mesenchymal nephrotoxicity poses a major limitation of drug therapy. On the one hand, reductions by 33% of normal renal function in patients treated with CsA for autoimmune disease appear to be reversible on discontinuance of therapy. On the other hand, severe renal dysfunction occurs in transplant recipients, particularly in the presence of concomitant therapy with nephrotoxic drugs. In the kidney transplant setting, numerous factors exacerbate the druginduced injury, including adverse donor procurement conditions, prolonged graft storage, and allograft rejection. Since the clinical and histologic findings in CsA nephrotoxicity tend to be pleiomorphic, no single diagnostic or therapeutic strategy has emerged. However, insights gained through pharmacologic monitoring of renal allograft recipients suggest that the group at increased risk for nephrotoxicity can be identified by elevated serum drug trough levels, increased area under the plasma time concentration curve, prolonged elimination half-life, and heightened pharmacodynamic sensitivity to CsA. Analysis of pharmacologic data in conjunction with clinical results should provide drug regimens of maximal therapeutic index with minimal risk of complications.
The quantitative analysis of phospholipid molecular species containing polyenoic fatty acids is described. Dinitrobenzoyl derivatives of diacylglycerols prepared from phospholipids were separated into individual molecular species by reversed-phase high performance liquid chromatography (HPLC) using a combination of two solvent systems and were quantified at 254 nm. Thirty-six molecular species were resolved from the phosphatidylcholines of rat hearts, human platelets and Chinese hamster V79-R cells. The derivatives of alkenylacyl molecular species from platelet phosphatidylethanolamine were resolved concomitantly with diacyl molecular species.
Mouse spleen cells were separated into Ig+ and Ig- populations by positive selection using petri plates coated with rabbit anti-mouse immunoglobulin. The Ig- cells were subsequently incubated with mouse monoclonal alloantisera to Thy1.2 prior to a second positive selection. The adherent populations were characterized as B (Ig+) or T (Thy1.2+) lymphocytes on the basis of surface immunofluorescence and mitogen-induced proliferation. Analysis of the 2 isolated populations by flow cytometry showed that B and T lymphocytes could be distinguished by their forward light scatter as well as their fluorescence after incubation with fluorescein diacetate.
Studies were performed to further characterize the effects of saturated fatty acids on murine T lymphocyte proliferation. Flow cytometry was used to show that the inhibitory effects of stearic acid (18:0) on [3H]thymidine uptake can be correlated with changes in cellular DNA content. Additional studies using flow cytometry and fluorescein diacetate as a viability stain showed that exogenous 18:0 was toxic for phytohemagglutinin (PHA)-stimulated T cells, whereas the viability of unstimulated T cells was less affected by 18:0. The inhibitory effects of 18:0 on T cell proliferation were evident as early as 4 hr after fatty acid addition and after a 10-hr exposure, the effects of 18:0 could not be reversed by washing the cells or by adding oleic acid (18:1). It is proposed that the inhibitory effects of 18:0 are dependent upon PHA-induced changes in T cell lipid metabolism.
Saturated and unsaturated long-chain fatty acids were compared for their effects on mitogen-induced DNA synthesis in mouse B and T lymphocytes. At high concentrations (100-120 microM) all of the fatty acids tested were inhibitory to some extent, while at lower concentrations (20-60 microM) only the saturated fatty acids suppressed DNA synthesis. The inhibitory effects of the saturated acids could be reversed by the simultaneous addition of either a cis- or a trans-monounsaturated or a cis,cis-diunsaturated fatty acid. Compared to T cells stimulated with either phytohaemagglutinin or concanavalin A, B cells stimulated with either lipopolysaccharide or 8-bromoguanosine were considerably less susceptible to the inhibitory effects of the saturated fatty acids. These results demonstrate that free fatty acids may be useful tools for delineating the metabolic events involved in B cell and T cell activation.
Using the phosphatidylcholine specific transfer protein from bovine liver, native phosphatidylcholine from intact human erythrocytes was replaced by a variety of different phosphatidylcholine species without altering the original phospholipid and cholesterol content. The replacement of native phosphatidylcholine by the disaturated species, 1,2-dipalmitoyl- and 1,2-distearoylphosphatidylcholine, proceeded at a low rate and extensive replacement could only be achieved by repeatedly adding fresh donor vesicles. The replacement by disaturated molecules was accompanied by a gradual increase in osmotic fragility of the cells, finally resulting in hemolysis when 40% of the native PC had been replaced. Up to this lytic concentration, the replacement did not affect the permeability of the membrane for potassium ions. Essentially, all of the PC in the outer monolayer of the membrane could be replaced by 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine. These replacements did not alter the osmotic fragility of the cells, nor the K+ permeability of the membrane. Increasing the total degree of unsaturation of the phosphatidylcholine species modified the properties of the membrane considerably. Replacement by 1,2-dilinoleoylphosphatidylcholine resulted in a progressive increase in osmotic fragility and hemolysis started to occur after 30% of the native PC had been replaced by this species. K+ permeability was found to be slightly increased in this case. Cells became leaky for K+ upon the introduction of 1-palmitoyl-2-arachidonoylphosphatidylcholine in the membrane. The increased permeability was also reflected by an apparent increase in the resistance of the cells against osmotic shock. The conclusions to be drawn are that (i) 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine are species which fit most optimally into the erythrocyte membrane; (ii) loss of membrane stability results from an increase in the degree of saturation of phosphatidylcholine (unsaturation index greater than 0.5) and (iii) the permeability is enhanced by increasing the content of highly unsaturated species (unsaturation index greater than 1.0).
Vesicles of a variety of types of phosphatidylcholine have been shown to be suitable donors of phosphatidylcholine to intact rat erythrocytes in the presence of a specific phosphatidylcholine exchange protein. Coincident with the progression of phosphatidylcholine exchange is the onset of hemolysis, occurring at degrees of exchange characteristic for the type of phosphatidylcholine employed. During exchange with the dimyristoyl, dipalmitoyl and distearoyl species, hemolysis starts when 27%, 25% and 22% of the native phosphatidylcholine is replaced by these disaturated species, respectively. In the case of dielaidoylglycerophosphocholine and 1-stearoyl-2-oleoylglycerophosphocholine hemolysis starts after the introduction of 30% and 28%, respectively. In contrast, the replacement of native erythrocyte phosphatidylcholine with egg phosphatidylcholine, and the dioleoyl species up to levels of 60% does not result in rapid hemolysis. Despite such functional consequences, overall contents of the individual phospholipids and cholesterol are normal. Accompanying the biochemical events are morphologic changes, including echinocyte an spherocyte formation. The structure-function implications and possible mechanisms of hemolysis are discussed.
When low concentrations (2-5 mole %) of cis unsaturated free fatty acids (group A) are intercalated into lymphocyte plasma membrane, capping is inhibited. No effect is seen with trans unsaturated or saturated fatty acids (group B). The capping inhibition is reversible with increasing doses of extracellular calcium. Fluorescence photobleaching recovery has shown that the group A free fatty acids do not inhibit the receptor immobilization associated with patch formation, but inhibit the final energy-dependent movement of the patched receptors into a cap. We have also shown that the group A free fatty acids cause a shift in membrane-bound calcium to the lipid phase from probable protein-associated sites. We have incorporated these findings into a model for capping and membrane-cytoskeletal interactions.
Phosphatidylcholine of resting human tonsil lymphocytes contained dipalmitoyl (18 mol%), 1-oleoyl,2-palmitoyl (11 mol%) and dioleoyl (5 mol%) species. 1-Oleoyl and 1-linoleoyl species were also detected in other more highly unsaturated phosphatidylcholine species. The features of phospholipid synthesis in lymphocytes were investigated by incubating cells with radiolabeled precursors. The de novo synthesis of phospholipids occurring in lymphocytes was estimated to be physiologically important, in particular for supplying dipalmitoylglycerophosphocholine and highly unsaturated phosphatidylethanolamine. It was also suggested that diacylglycerol(s) not originating from glycerophosphate is (are) involved in the synthesis of tetraenoic phospholipids. From radioactive palmitic and oleic acids were actively synthesized dipalmitoyl, dioleoyl and 1-oleoyl,2-palmitoyl species of diacylglycerol. The mode of diacylglycerol synthesis was reflected upon phosphatidylcholine formation. The formed polyunsaturated phosphatidylethanolamine was also found to contain 1-oleoyl or 2-palmitoyl species. Radioactive linoleic and arachidonic acids were incorporated predominantly into the C-1 position of diacylglycerol, whereas the majority of the formed phospholipids was of 2-linoleoyl or 2-arachidonoyl species. Labeled stearic acid was exclusively esterified to the C-1 position of the glycerolipids. However, the labeling pattern of molecular species by stearate was considerably deviated from that observed with labeled palmitate. These results indicate that the de novo synthetic pathway operating lymphocytes is primarily responsible for forming 1-unsaturated type of phospholipids. The synthesis of 1-saturated,2-unsaturated species appeared to be due to remodeling of once-formed phospholipids.