Cell activation state influences the modulation of HLA-DR surface expression on human monocytes/macrophages by parenteral fish oil lipid emulsion

El estado de activación celular influye en la modulación de la expresión del HLA-DR en la superficie de los monocitos/macrófagos humanos mediante una emulsión lipídica parenteral de aceite de pescado

R. S. Torrinhas, T. Jacintho, H. Goto, M. Gidlund, M. M. Sales, P. A. Oliveira and D. L. Waitzberg

University of São Paulo Medical School. São Paulo. Brazil.

The authors thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP- 98/11379-9 and 99/08332-3) for financial support and Fresenius Kabi for kindly providing the parenteral lipid emulsions.

Correspondence

ABSTRACT

Abnormal surface expression of HLA-DR by leukocytes is associated with a poor prognosis in critical care patients. Critical care patients often receive total parenteral nutrition with lipid emulsion (LE). In this study we evaluated the influence of fish oil LE (FO) on human monocyte/macrophage (Mf) expression of surface HLA-DR under distinct activation states. Mononuclear leukocytes from the peripheral blood of healthy volunteers (n = 18) were cultured for 24 hours without LE (control) or with 3 different concentrations (0.1, 0.25, and 0.5%) of the follow LE: a) pure FO b) FO in association (1:1 - v/v) with LE composed of 50% mediumchain trygliceride and 50% soybean oil (MCTSO), and c) pure MCTSO. The leukocytes were also submitted to different cell activation states, as determinate by INF-γ addition time: no INF-γ addition, 18 hours before, or at the time of LE addition. HLA-DR expression on Mf surface was evaluated by flow cytometry using specific monoclonal antibodies. In relation to controls (for 0.1%, 0.25%, and 0.5%: 100) FO decreased the expression of HLA-DR when added alone [in simultaneously-activated Mf, for 0.1%: 70 (59 ± 73); for 0.25%: 51 (48 ± 56); and for 0.5%: 52.5 (50 ± 58)] or in association with MCTSO [in simultaneously-activated Mf, for 0.1%: 50.5 (47 ± 61); for 25%: 49 (45 ± 52); and for 0.5%: 51 (44 ± 54) and in previously-activated Mf, for 1.0%: 63 (44 ± 88); for 0.25%: 70 (41 ± 88); and for 0.5%: 59.5 (39 ± 79)] in culture medium (Friedman p < 0.05). In relation to controls (for 0.1%, 0.25%, and 0.5%: 100), FO did not influence the expression of these molecules on non-activated Mf [for 0.1%: 87.5 (75 ± 93); for 0.25%: 111 (98 ± 118); and for 0.5%: 101.5 (84 ± 113)]. Results show that parenteral FO modulates the expression of HLA-DR on human Mf surface accordingly to leukocyte activation state. Further clinical studies evaluating the ideal moment of fish oil LE infusion to modulate leukocyte functions may contribute to a better understanding of its immune modulatory properties.

Key words: Parenteral fat emulsions. HLA-DR. Fish oil.

RESUMEN

La expresión anormal del HLA-DR en la superficie de los leucocitos se asocia con un pronóstico peor en los enfermos críticos. Estos enfermos a menudo reciben nutrición parenteral total con una emulsión lipídica (EL.) En este estudio evaluamos la influencia de la EL de aceite de pescado (AP) sobre la expresión del HLA-DR de superficie por los monocitos /macrófagos humanos (Mf) en distintos estados de activación. Se cultivaron leucocitos mononucleares de sangre periférica de voluntarios sanos (n = 18) durante 24 horas sin EL (control) o con tres concentraciones diferentes (0,1, 0,25 y 0,5%) de la siguiente EL: a) AP puro b) AP en asociación (1:1 en v/v) con la EL compuesta de un 50% de triglicéridos de cadena media y 50% de aceite de soja (TCMAS), y c) TCMAS puro. Se sometió a los leucocitos a tres estados de activación diferentes, como venía determinado por el tiempo de adición de INF-γ: sin añadir INF-γ, 18 horas antes o en el momento de añadir la EL. La expresión de HLA-DR en la superficie de los Mf se evaluó mediante citometría de flujo empleando anticuerpos monoclonales específicos. En relación con los controles (para 0,1%, 0,25% y 0,5%: 100) el AP disminuyó la expresión de HLA-DR cuando se añadía solo {en Mf activados de forma simultánea, para 0,1%: 70 (59 ± 73); para 0,25%: 51 (48 ± 56) y para 0,5%: 52,5 (50 ± 58)} o en asociación con TCMAS [en Mf activados de forma simultánea, para 0,1%: 50,5 (47 ± 61); para 25%: 49 (45 ± 52); y para 0,5%: 51 (44 ± 54) y en Mfactivados previamente, para 1,0%: 63 (44 ± 88); para 0,25%: 70 (41 ± 88); y para 0,5%: 59,5 (39 ± 79)] en medio de cultivo (Friedman p < 0,05.) En relación con los controles (para 0,1%, 0,25% y 0,5%: 100), el AP no influyó en la expresión de estas moléculas en los Mfno activados [para 0,1%: 87,5 (75 ± Ó93); para 0,25%: 111 (98 ± 118); y para 0,5%: 101,5 (84 ± 113)}. Los resultados muestran que el AP parenteral modula la expresión del HLA-DR sobre la superficie de los Mfhumanos en función del estado de activación de los leucocitos. Estudios clínicos adicionales que evalúen el momento ideal de la infusión de la EL con aceite de pescado para modular las funciones leucocitarias podrían contribuir a un mejor conocimiento de sus propiedades inmunomoduladoras.

Palabras clave: Emulsiones grasas parenterales. HLA-DR. Aceite de pescado.

Introduction

Monocytes/macrophages actively participate in the innate and acquired immune responses against foreign antigens. Human leukocyte antigen (HLA) molecules from the major complex of histocompatibility class II (MHC II) system expressed on monocytes/macrophages surface represent the link between innate and acquired immunity, playing a central role in activation of the cell-mediated immune response.¹¹

Abnormal expression of HLA-DR is associated with a poor prognosis in several clinical conditions. For instance, increased surface expression of HLA-DR in activated monocytes/macrophages is seen in patients with rheumatoid arthritis and is associated with stronger activation of their inflammatory response and worsening of their clinical condition.¹² On the other hand, a marked decrease of surface HLA-DR in monocytes can be observed after massive hyper-inflammatory reactions and is associated with functional deactivation of monocytes and poor prognosis in sepsis. ^13,14

Taken together, these observations suggest that surface expression of HLA-DR on monocytes/macrophages is strongly dependent on the activation state of these leukocytes. Increment or decrement of such expression may bedesirable, according to the patient's clinical condition.

The use of FO has been proposed in critical care patients in attempt to attenuate inflammation. It has been demonstrated clinically that the FO infusion in these patients is associated with decreased production of inflammatory cytokines.^9-10 From a mechanistic point of view, it is of interest to study the modification of a central immune signaling molecule under different stress states via the use of FO.

The aim of the present study was to evaluate the effect of FO on surface expression of HLA-DR by human monocyte/macrophage at different activations states.

Methods

Subjects

After local ethical committee (Comissão de Ética para Análise de Projetos de Pesquisa-CAPPesq) approval, heparinized blood samples were drawn from healthy adult (20-40 year old) male volunteers (n = 18) selected by a questionnaire. The questionnaire excluded smokers, athletes, alcoholic, drugs users, and illness up 3 weeks prior to blood collection.

Mononuclear leukocytes were isolated from whole blood by Ficoll-Hypaque (Histopaque^® 1077, Sigma-Aldrich-USA) density gradient centrifugation, according to the modified Boyum technique.¹⁵ Briefly, peripheral blood from each donor (usually 40 mL) was collected into heparinized vacuum tubes (Vacutainer^®, Becton-Dickinson-USA), diluted (1:1) in saline (Baxter-USA), added to 50 mL tubes (BD Falcon™-USA) containing Ficoll-Hypaque (2:1) and centrifuged (Eppendorf 5810R-USA) for 30 minutes at 2000 rpm and 18^oC. Mononuclear cells at the interface were aspirated and washed twice with equal volume of phosphate buffered saline (PBS) pH 7.2 (Sigma-USA), to be further cultured with different LE.

Mononuclear leukocyte culture with LE

After mononuclear leukocyte separation, the cells were plated under sterile conditions in 24 wells plates (Costar-USA), 2x10⁶ cells per well, and cultured with or without 4 x 10⁵ U/L of INF-γ (Genzyme - USA). According to the moment of INF-γ addition to the culture, mononuclear leukocytes were considered to have 3 different activation states: non-activated: without INF-γ addition; previously-activated: with INF-γ addition 18 hours before LE addition; and simultaneouslyactivated: with INF-γ addition at the same time of LE. LE were diluted at 0.1%, 0.25%, and 0.5% concentrations in 2 mL of HEPES buffer RPMI medium (RPMI 1640, Gibco-USA), containing 5% heat-inactivated fetal calf serum (Gibco-USA), 1x10⁵ U/L penicillin (Sigma-USA), and 0.07 mmol/L gentamicin (Sigma-USA).

According to the type of LE added in non-activated, previously activated or simultaneously activated mononuclear leukocytes, there were 4 experimental groups: C-control without LE; MCT - LE composed of a physical mixture of 50% medium chain triglycerides and 50% soybean oil (Lipovenos^® MCT 20%, Fresenius-Kabi-Germany); FO-fish oil LE (Omegavenos^® 10%, Fresenius-Kabi-Germany); and MCTFO-LE composed of an experimental mixture of the LE composed by a physical mixture of medium chain triglycerides and soybean oil with FO (1:1 v/v). Table I describes the usual compositions of all LE.

During the entire culture period, mononuclear leukocytes were kept in a moist atmosphere at 37^oC in a 4% CO2 incubator (Revco Elite, Revco Technologies-USA). The mononuclear leukocytes were found to be > 90% viable, as accessed by Trypan Blue (Sigma-USA) exclusion.

Immunofluorescence staining

After LE culture, the leukocytes were washed twice with PBS and incubated in a dark room at 4^oC for 30 minutes with 10 μL of AB serum and 10 μL of the following monoclonal antibodies: anti-HLA-DR stained with phycoerythrin (PE) and anti-CD14 stained with allophycocyanin (APC), all from BD Pharmigen-USA. Nonspecific binding was corrected by using cells stained with the appropriate isotype-matched immunoglobulin (Ig) controls (BD Pharmigen-USA). After incubation, mononuclear leukocytes were washed twice with 2 μL of PBS and fixed with 250 μL of 1% paraformaldeyde (Sigma-USA) solution immediately before flow cytometry acquisition.

Flow cytometry analysis

Statistical analysis

The mean of data from fluorescence intensity samples was converted to percentage of the basal expression determined by the control group (considered as 100%). Friedman test followed by the Student-Newman-Keuls post hoc test were applied to compare differences across groups using SigmaStat software (Sigma-EUA). P ≤ 0.05 was considered statistically significant.

Results

FO did not influence surface expression of HLA-DR on non-activated monocytes/macrophages. In the MCT group, 0.1% lipid emulsion concentration decreased HLA-DR expression on non-activated monocytes/macrophages (P = 0.0042, table II).

For previously-activated monocytes/macrophages, FO combined with LE rich in medium-chain triglycerides at all concentrations decreased expression of HLA-DR compared to controls without LE (P = 0.019, table II).

For simultaneously-activated monocytes/macrophages, FO alone at all concentrations with significant doses dependence (P = 0.004, table II) and also when associated with LE rich in medium-chain triglycerides at all concentrations (P = 0.007, table II) decreased HLADR expression compared to control. This inhibitory effect was highest in the MCTFO group with an LE concentration of 0.10% (P = 0.006, table II). Both 0.25% (P = 0.0003) and 0.50% (P < 0.0001) concentrations of FO alone or combined with LE rich in mediumchain triglycerides decreased the expression of HLADR (table II).

In order to simulate the environment of the blood stream during parenteral infusion of lipid emulsions, our in vitro experimental model was designed considering the culture of total mononuclear cells instead of just monocytes/macrophages, because leukocyte interactions that occur in in vivo may influence their response to external stimuli.¹⁶.

The reported physiological concentration of LE in leukocyte cultures varies widely. In order to determine the ideal concentration of LE for our experimental model, we conducted a pilot study and tested cell viability using trypan blue dye exclusion in monocytes/macrophages cultures ranging 0.1-1% of lipid emulsion in culture medium. Cultures with 1% FO in both previously and simultaneously activated monocytes/macrophages were associated with less than 80% cell viability, probably due to excessive production of free radicals associated with polyunsaturated FA combined with those provided by leukocyte reaction after INFgamma stimuli.^17,18 Thus the 1% concentration of FO was excluded.

We compared the effect of pure FO on HLA-DR versus LE composed of 50% medium chain triglyceride and 50% soybean oil (MCT/LCT) because the latter is largely used in clinical practice and is associated with few effects on immune function.^19,20 Considering that FO is infused in combination with other conventional LE (soybean oil LE, MCT/LCT, or LE composed of 80% olive oil and 20% soybean oil) in clinical practice, we also compared the effect of pure FO with a experimental mixture composed of a high concentration of this LE with MCT/LCT (1:1).¹⁹

INF-γ was chosen as an external stimuli to activate human monocytes/macrophages because this cytokine up-regulates the surface expression of HLA-DR molecules on mononuclear leukocytes.²¹

Our results show that FO influences HLA-DR surface expression by monocytes/macrophages according to the leukocyte activation state. FO alone and mainly when combined at high doses with MCTSO (1:1) decreased surface expression of HLA-DR on simultaneously or previously-activated monocytes/macrophages but had no effect on leukocytes that were not activated.

Despite methodological differences, the improved effects observed with the mixture of FO and MCTSO versus pure FO were also observed in our previous studies. These studies showed the FO-MCTSO combination to increase the favorable effect of FO on eicosanoid production in a experimental model of colitis and also to favorably effect the number of spleen resident, non-opsonized carbon particle phagocytingmacrophages in rats.^22,4 The stronger effect of the FOMCTSO mixture when compared to pure FO may be related to improved use of omega-3 FA by monocytes/macrophages. While omega-3 FA from FO are poor substrates for lipoprotein lipase (LPL), parenteral medium chain triglycerides (MCT) are more quickly degraded by this enzyme and are also a quick source of energy. Experimental studies showed that when omega-3 FA are infused with MCT, they are more easily released for cellular use.²³

In fact, our findings may be a result of the fatty acids EPA and DHA, which are provided by FO. In another experimental study, the in vitro effect of EPA and DHA on HLA-DR expression was evaluated in non-activated monocytes/macrophages and INF-γ-activated human monocytes/macrophages, where INF-γ stimulation occurred at the same time as EPA and DHA addition, corresponding to the simultaneously activated group of our study.²⁴ In agreement with our findings, EPA and DHA did not change HLA-DR expression on human non-activated monocytes but decreased the expression of these molecules on INF-γ activated monocytes and also decreased their ability to present antigen to autologous lymphocytes.²⁴

A reduction in antigen-presenting function may impair T cell activation, thus decreasing both inflammatory cytokine and B cell production. It was shown that cell activation state can influence the immunomodulatory effect of fish oil on cytokine production.²⁵ Mice fed fish oil for 6 weeks showed decreased ex vivo production of TNF-α and IL-1β by inflammatory macrophages (induced by intraperitoneal injection of thioglycollate broth 4 d prior to sacrifice) but not by non-inflammatory resident macrophages, which increased TNF-α production.²⁵ Despite methodological discrepancies, these findings are in agreement with the present study in demonstrating the inhibitory effect of fish oil on immune variables only under a cell activation stimulus. In addition, our data may also corroborate to suggest a possible reduction of antigen-presenting function through HLA-DR inhibition by fish oil as a possible mechanism related to the observed decrease in inflammatory cytokine production.

Despite methodological differences, our findings are also in accordance with other experimental studies that found an inhibition of la molecules (the mice equivalent of HLA-DR molecules) by fish oil. In mice genetically modified to develop autoimmune lupus (MRL-lpr mice) the ingestion of fish oil was associated with disease suppression and decreased Ia expression.²⁶ Mice and rats fed fish oil via esophageal gavage showed a reduced percentage of peritoneal macrophages expressing la.²⁷ Listeria monocytogenes-infected mice fed with fish oil had decreased expression of la on the surface of peritoneal macrophages in relation to mice fed with other fat sources.²⁸ Similar to our observation of HLA-DR inhibition by FO in monocytes/macrophages under a stress stimulus, these other reports also had a stress factor present (disease, esophageal gavages, and induced infection)during or before the fish oil supply.

In addition, the decreased expression of HLA-DR on simultaneously or previously activated but not on nonactivated human monocytes/macrophages after FO culture suggests a potential benefit in the use this LE in hyperinflammatory conditions followed by an immunodeficiency state, as occurs in sepsis, severe trauma, and burns.¹⁴ Our experimental data suggest that FO could prevent excessive monocytes/macrophages activation without the impairment of functional surface molecule expression. The clinical application of these experimental data remains to be further elucidated.

There are several mechanisms potentially involved in the modulatory effect of FO on HLA-DR surface expression by simultaneously or previously activated monocytes/macrophages. They include incorporation of omega-3 polyunsaturated fatty acids into the cell membrane, modification of lipid rafts, modulation of eicosanoids production, and modulation of gene expression for inflammatory mediators or surface molecules. ^32-37 While each of these proposed mechanisms has support in the scientific literature, they were not evaluated in the present study and require further analysis.

Taken together, our findings allow us to suggest that leukocyte activation state may be responsible for the overall disparate data regarding the in vitro effects of FO on immune function. Previously, these discrepancies have been attributed mainly to methodological variables between the scientific reports, such as differences in the cell culture conditions.

Regarding the substantial limitations to extrapolation of experimental in vitro results to clinical application, our findings also suggest that the patient's clinical condition may be crucial in determining the immune modulatory effect of FO.

Conclusion

Fish oil lipid emulsion, mainly when associated with lipid emulsion rich in medium-chain triglycerides, distinctly influences surface expression of HLA-DR on activated monocyte/macrophages. The inhibition of HLA-DR by FO may be protective in conditions where monocytes/macrophages are in constant activation.

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Correspondence: ]]> Raquel Susana Matos de Miranda Torrinhas.
University of São Paulo Medical School.
Av. Dr. Arnaldo 455, 2^oandar - sala 2208.
CEP 01246-903 Cerqueira César, São Paulo (SP). Brazil.
E-mail: metanutri@usp.br

Recibido: 22-I-2010.
Aceptado: 20-II-2010.