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Annals of Clinical & Laboratory Science 31:75-84 (2001)
© 2001 Association of Clinical Scientists

Expression and Secretion of RANTES by Human Peripheral Blood CD4+ Cells are Dependent on the Presence of Monocytes

Pio Conti1, Renato C. Barbacane1, Claudio Feliciani2 and Marcella Reale1
Department of Oncology and Neurosciences, University of Chieti School of Medicine, Chieti, Italy 1 Immunology Division; 2 Dermatology Section

Address correspondence to Professor Pio Conti, Immunology Division, University of Chieti School of Medicine, Via dei Vestini, 66013 Chieti, Italy; tel 39 0871 3555293; fax 39 0871 561635; e-mail pconti{at}unich.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The cDNA for RANTES (an acronym for "Regulated upon Activation, Normal T cell Expressed and Secreted") was initially discovered by subtractive hybridization as a T cell–specific sequence. Consistent with it being a C-C (ß) chemokine, RANTES is a monocyte chemoattractant. In addition, RANTES can chemoattract unstimulated CD4+/CD45RO+ memory T cells and stimulated CD4+ and CD8+ T cells with the naive and memory phenotypes in immunologically active sites. It has been shown that CD8+ cells are dominant sources of RANTES. Here we attempted to determine if CD4+ cells express and secrete RANTES alone, or if other accessory cells (activated monocytes) are needed to activate them. We found that when autologous monocytes are added to CD4+ cells and then stimulated with phytohaemagglutinin (PHA), the quantity of RANTES, in terms of transcription and translation of the protein, is significantly higher than the amount produced by the PHA–activated monocytes alone; isolated CD4+ cells stimulated with PHA do not produce any appreciable quantity of RANTES. When CD4+ cells are primed overnight with monocytes and then stimulated with PHA, they produce more RANTES compared to PHA–stimulated CD4+ cells alone. The influence that monocytes have on CD4+ cells to produce RANTES was confirmed when the physiological activator, tumor necrosis factor- {alpha} (TNF-{alpha}), was used. These data show that CD4+ cells need monocytes to express and secrete appreciable amounts of RANTES.

(received 10 August 2000; accepted 6 October 2000)

Keywords: RANTES, CD4+ cells, monocytes, macrophages, dexamethasone


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
RANTES (an acronym for "Regulated upon Activation, Normal T cell Expressed and Secreted") and other chemoattractant proteins are members of the intercrine or chemokine family of proinflammatory basic polypeptides [1]. RANTES is a proto-type of the C-C chemokine subfamily that acts as a selective chemoattractant of human monocytes and CD4-positive lymphocytes [2,3] and increases the adherence of monocytes to endothelial cells [4]. RANTES causes mast cell recruitment and increases transcription of histidine decarboxylase in mice [5–7].

The human CD4 and CD8 phenotypes were first described many years ago, but many of the physiological functions of each population and their leukocyte-leukocyte interactions have not been completely characterized. In general, CD4+ cells function as helper cells; however, the functional role of these accessory cells might be particularly important when they participate as trigger activators in the inflammatory response. When activated, CD4 T cells modulate the level and direction of immune responses through the release of cytokines; CD4 cells may provide additional signals to other lymphocyte subsets to help amplify and direct immune response. These important signals may include the highly bioactive inflammatory molecules termed chemokines. Chemokines recruit and accumulate immune cells during an immune reaction. Accumulating evidence implicates the infiltrating mononuclear phagocytes in orchestrating immune and inflammatory responses through their ability to produce multiple regulatory factors. The nature of the cell populations homing to immunologically active sites varies according to the type of immune response. The selective chemoattractant and pro-adhesive effects of chemokines identify them as candidates to play a key role in lymphocyte trafficking in many diseases [8].

Recently, it has been reported that T helper 1 and 2 CD4+ cells respond differentially to chemokines, and that the expression of chemokine receptors may be extremely important in determining the sensitivity of T cells to antigen activation [9,10]. RANTES is an ideal candidate for recruitment of specific effector cells to inflammatory sites or other lesions because of its selective chemoattractant activities [11,12]. Although the expression of RANTES was initially thought to be limited to activated T cells, evidence shows that it is produced by a variety of cells in response to stimuli [13,14]. In this study we analyzed the human CD4 response to PHA, with or without monocytes, with respect to RANTES mRNA synthesis and protein secretion. Interestingly, the transcription and translation of the RANTES protein is associated with the presence of activated monocytes. This leads us to suggest that RANTES is a principal mediator in inflammatory processes, where CD4 and monocytes are specifically required. We demonstrate that CD4+ cells and monocytes differ in their intrinsic response properties and their RANTES production in response to nonspecific antigens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Isolation of human peripheral blood mononuclear cells.  Peripheral blood mononuclear cells (PBMC) were collected from healthy adult donors and anticoagulated with heparin (20 U/ml). Blood was diluted 1:1 with saline solution, underlayered with Ficoll-Hypaque (Sigma, St Louis, MO, USA), and centrifuged (800 x G, 30 min). Mononuclear cells were washed 3 times with phosphate-buffered saline (PBS). Cell viability was examined by the trypan blue exclusion method.

Monocyte purification.  Aliquots of PBMC were incubated at 10 x 106 cells/ml in RPMI 1640 (Sigma) supplemented with 10% fetal calf serum ( Sigma) for 1 hr in plastic tissue culture flasks ( Corning T-75-cm2, Corning, NY) at 37°C in a 5% CO2 incubator. The cells were allowed to adhere for one hr, and not more, to avoid further activation. After that time the supernatants, which contained nonadherent cells, were discarded and the remaining adherent monocytes (about 10% of the original population) were removed from the flask by vigorous washing with ice-cold phosphate-buffered saline (PBS) and scraping of the flask. The isolated cells were washed and their viability tested. The cell suspension consisted of 90% monocytic leucocytes (CD14+, as analyzed by flow cytometry); neutrophils and platelets were approximately 10%. The culture medium contained less than 10 pg/ml of lipopolysaccharide (LPS), as determined by the limulus amoebocyte test (E-Toxate, Sigma).

Immunomagnetic negative selection of CD4+ cells.  Nonadherent cells obtained as described above were rosetted with monosized superparamagnetic polystyrene beads (Dynabeads M-450, Dynal, Oslo, Norway), according to the manufacturer’s instructions, and coupled with a primary monoclonal antibody (mAb) specific for the CD8 membrane antigen [15]. The mouse IgM mAb was directly adsorbed onto the Dynabeads’ surface. The mouse IgM mAb (ITI-5C2) employed recognizes the 32kDa CD8 antigen. The epitope recognized by the ITI-5C2 mAb is closely related to the Leu2a epitope that resides on the CD8{alpha}-chain.,

Dynabeads with monoclonal antibody and target cell suspension (70 µl of beads; 107 cells/ml) were mixed in an ice bath that provided both gentle tilting and rotation. After incubation for 30 min, the CD8 positive T cells had become bound to Dynabeads’CD8. Then the volume was increased by adding PBS with 1% FCS to reach a final volume of 10 ml, and the suspension was exposed to a magnetic field to separate the rosetted cells and unbound Dynabeads. The CD8-depleted cell suspension was transferred to a second tube and the washing was repeated at least twice. The isolated cells were CD45RO+ cells (> 95%) and viable (> 95%), while human CD45RA+, as revealed by Ab FITC (Sigma, code F1527), were practically nil. For purification of CD4 cells, we chose an appropriate monoclonal (MAb) (10 x MAb) mix including CD14, CD16, CD20, and antiglycophorin, and added MAb specific for CD8. A CD11b MAb was added to bind the subset of CD8+ cells, as well as monocytes, B cells, and natural killer cells, to guarantee depletion of those contaminant cells. Then, CD45RO microbeads (2 ml, 109 total cells) (Miltenyi Biotec Inc, Auburn, CA, USA) were employed for positive selection of T-cell subsets from peripheral blood. Cells isolated with CD45RO microbeads were used in this study and referred to simply as CD4+. Virtually no naive T cells were found in the CD4 population using CD45 Ab FITC (Sigma, code F1527). In contrast, memory cells were selected, as shown by expression of CD45RO+ (CD45RA-).

Induction of RANTES synthesis in monocytes and CD4+ T cells.  Dexamethasone is a potent inhibitor of cellular metabolism and arachidonic acid formation, and also decreases mRNA stability of molecules involved in inflammation (eg, IL-1, IL-8). Adherent cells, PBMC, CD4+, and CD4+CD8 T cells were incubated overnight in the absence or presence of dexamethasone (DXM) 10-7 M. The next day the cells were washed twice and cultured in RPMI 1640 with 10% fetal calf serum (FCS) at 2 x 106 cells/ml.

In some experiments, CD4+ cells (1.5 x 106/ml ), treated or not with DXM, were cultured with adherent, autologous cells (0.5 x 106/ml) that were untreated or treated with DXM (10-7 M). Cells were cultured for 18 hr with PHA (20 µg/ml) (Sigma) and TNF{alpha} (10 ng/ml) (Peprotech, Milan, Italy), and then centrifuged; the pellets were used for RNA extraction and the supernatants stored at -80°C until examination for RANTES by ELISA assay (ELISA, R&D Systems, Minneapolis, MN, USA). We chose tumor necrosis factor-{alpha} (TNF{alpha}) for its ability to regulate chemokine production [16]. We used PHA because it is a good stimulator for antigenic chemokines [17].

Quantification of RANTES.  Antigenic RANTES was quantified using a solid phase, sandwich-type, enzyme-linked immunoassorbent assay (ELISA, R&D Systems, Minneapolis, MN, USA.) according to the manufacturer’s recommended procedures. A monoclonal antibody specific for RANTES was coated onto the wells of flat-bottomed 96-well microtiter plates. Standards or samples were added; during the first incubation the RANTES antigen bound to the immobilized antibody on one site. During the second incubation, the biotinylated polyclonal antibody bound to the immobilized RANTES captured during the first incubation, after streptavidin-peroxidase was added, and after a third incubation a substrate solution was added. The intensity of the colored product is proportional to the concentration of RANTES present in the sample.

Dexamethasone treatments.  Dexamethasone (Merck, Sharp, & Dohme, Rome, Italy) was always used at 10-7 M. Dexamethasone-treated monocytes and CD4 cells were washed out after an overnight incubation and then the cells were added to the culture, stimulated or not with PHA or TNF{alpha}. This concentration proved to be non-toxic—it was used in cell cultures in our laboratory for extended periods (over 30 days)—however, cell viability was always checked after each drug treatment.

Northern blot analysis.  Northern blot analysis was performed on purified cells after the culture, tested with hrRANTES (20 ng/50 µg), the negative control (PBS) and the positive control (LPS), and anti-RANTES-Ab (50 µg/mouse) (R. & D Systems, Inc., Minneapolis, MN, USA). Total RNA was isolated with guanidine hydrochloride as previously described [18]. Total RNA (10 µg/lane) was fractionated by electrophoresis on a formaldehyde denaturing agarose gel, transferred to nylon membranes (Hybond N, Amersham Ltd, Amersham, UK) and hybridized with 32P labelled HDC cDNA probe (2 x 108 cpm/mg). It was then washed 4 times at room temperature for 15 min in a 2x solution of sodium citrate (SSC) and 0.1% SDS, heated to 48°C for 30 min, and then washed twice in 0.1x SSC and 0.1% SDS. Finally, membranes were exposed to Kodak XAR5 for 3 days at -70°C. Signals were compared with ribosomal RNA to evaluate an equal quantity of RNA for each lane. Densitometric analysis was performed with a computerized image analyzer (Quantimed software, Leica, Heidel Northern blot analysis, Northerberg, Germany) for normalization of relative mRNA levels (as reported in Results).

mRNA extraction and cDNA synthesis.  Poly-A mRNA was extracted using a purification system kit (Pharmacia Biotech, Milan, Italy). In brief, about 10 million cells were dissolved in a solution containing guanidinium thiocyanate 4M and N-lauroylsarcosine in order to preserve the RNA. The solution was placed in oligo(dt)-cellulose at 25 µg/ml suspended in a storage buffer containing 0.15% Kathon CG (Pharmacia LKB, Cologno Monzese, Milan, Italy). After several washes in salt buffers containing 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.5 M NaCl, or 0.1 M NaCl in the last 2 washes, the oligo(dt)-cellulose containing mRNA was placed in filter columns and mRNA was eluted in warm TRIS-HCl 10 mM and precipitated in chilled 95% ethanol overnight. After centrifugation, the pellet was dissolved in 14 µl of DEPC-treated sterile water and quantitated by spectrophotometric analysis. The mRNA (0.5 µg) was transcribed in cDNA, incubating it with 200 U of superscript reverse transcriptase (GIBCO BRL, Milan, Italy) and 50 ng of random examers.

Reverse transcriptase PCR amplification.  cDNA was amplified with 2.5 U Taq polymerase (Perkin Elmer Cetus, Milan, Italy) using 1.5 pM of each primer specific for RANTES and G3PDH. RANTES and G3PDH primers were purchased from Clonetech Laboratory (Palo Alto, CA, USA). Each sample was divided in half; one half was used for the cytokine under investigation, the other half for the G3PDH for semiquantitative analysis. RT-PCR was conducted for RANTES with the following protocol: 1) predenaturation at 94°C for 5 min, 2) denaturation at 94°C for 2 min, 3) annealing at 55°C for 2 min, 4) extension at 72°C for 2 min, and 5) denaturation at 94°C for 2 min. After repeating steps 3 through 5 for 29 cycles for RANTES (and 25 cycles for G3PDH), re-extension was performed at 72°C for 5 min. Five ml of amplified products were electrophoretically separated in 2% agarose gel containing ethidium bromide and finally analyzed for molecular size. The following controls were used: cDNA without primers; normal muscle tissue. Signals were analyzed by Bio-profile software (Vilber Lourmat) and semiquantitative analysis was achieved by comparing the amplified product signals with the G3PDH signal.The sequences of the 5' sense primers and 3' antisense primers that were used are: RANTES, 5'-ATATTCCTCGGACACCACAC-3', and 5'-CACTCCAGCCTGGG GAAGG-3' (product size, 370 bp).

Statistical Analyses.  Data from 4 different experiments were combined and reported as the mean ± SD. The t-test for independent means was used for statistical analyses (p >0.05 was considered not significant).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
RANTES generation by PHA-activated peripheral blood mononucear cells.  RANTES has selective chemo-attractant properties for monocytes/macrophages, memory T cells (CD45RO/CD45RA-), and basophils, rather than neutrophils [1,2,19]. We wanted to demonstrate that activated monocytes, among other accessory cells, produce RANTES after treatment with PHA. Fig. 1Go shows the effect of PHA on RANTES production by activated human peripheral blood mononuclear cells, based on quadruplicate experiments. RANTES was measured by the ELISA method after overnight incubation. The treatment of PBMC with PHA produced elevated RANTES generation from an overnight exposure (2,646 ± 50 pg/ml), while the control (untreated cells) generated 220 ± 40 pg/ ml. The effect of PHA (20 µg/ml) on dexamethasone pretreated cells was similar to the effect on the control cells (106 ± 45 pg/ml).



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  		Fig. 1. RANTES production by PBMC (2 x 106/ml) activated, or not, with PHA (20 µg/ml) and overnight pretreated (18 hr), or not, with dexamethasone (10-7M) plus PHA. Experiments were performed in quadruplicate.

 
Increase of RANTES mRNA levels on PHA-activated PBMC.  In these studies we analyzed the generation of RANTES mRNA in PBMC cultures. Steady-state levels of RANTES mRNA in control were low (Fig. 2Go, lane 1). In the presence of PHA (lane 2), RANTES mRNA was higher compared with the controls. When the cells were exposed to DXM (lane 3) and then treated with PHA (20 µg/ml), the results were not different from those for the control. Quantative analysis by densitometer (Ultrascan XL, LKB-Pharmacia, Uppsala, Sweden) on mRNA expression reflected the findings shown in Fig. 2Go (data not shown).



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  		Fig. 2. Northern blot analysis of RANTES mRNA levels in PHA (20 µg/ml)-activated PBMC (2 x 106/ml), overnight pretreatment (18 hr), or not, with dexamethasone (10-7M). Dexamethasone strongly inhibited the PHA effect (lane 3). Representative results of those obtained in four independent experiments with cells from four different donors.

 
Generation of RANTES on CD4+ (CD45RO+) cells and/or monocytes activated with PHA.  RANTES in lymphocytes and monocytes play an important role in providing direct cellular contact (inducing adhesion molecules) and directing the migration of specific leukocytes. We investigated whether the presence of isolated monocytes would alter the response of PHA-stimulated purified CD4+ cells (Fig. 3Go). Monocytes and/or CD4 lymphocytes from the same donor were isolated and stimulated with PHA 20 µg/ml alone or in combination. After 18 hr of stimulation, cell-free supernatants were harvested and examined for the presence of RANTES by ELISA. As shown in Fig. 3Go, the addition of monocytes to CD4 cells (CD4 + Mø) increased the production of RANTES in cells exposed to PHA. However, RANTES generation from PHA-stimulated CD4 and dexamethasone-pretreated monocytes was not significant compared to the controls (unstimulated cells). Moreover, when CD4 cells were pretreated with dexamethasone and added to the purified monocyte population, the release of RANTES after PHA stimulation was significantly increased compared to the control, but was less than that seen in CD4 cells plus PHA-stimulated monocytes. When PHA-stimulated CD4 cells with monocytes were compared with PHA-stimulated monocytes alone, the difference was statistically significant .



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  		Fig. 3. Production of RANTES by CD4 cells, monocytes (Mø), CD4 cells + Mø, CD4 cells + Mø pretreated with DXM overnight, and CD4 cells pretreated with DXM overnight. The cells were cultured for 18 hr and stimulated, or not, with PHA (20 µg/ml). The overnight incubation with DXM (10-7M) was approximately 18 hr. The p values (t-test) were obtained comparing control (C) with PHA treatments (NS, not significant). When PHA-stimulated CD4 plus Mø was compared with PHA-stimulated Mø alone, the difference was significant (p < 0.05). Experiments were performed in quadruplicate.

 
RANTES mRNA detected on PHA-activated CD4 cells plus monocytes.  Because RANTES is chemotactic for different populations of inflammatory cells and is produced by different leukocytes, we postulated that CD4+ cells stimulated by PHA need accessory cells to express high levels of RANTES mRNA. We examined RANTES mRNA expression in purified CD4+ cells plus monocytes, CD4+ plus dexamethasone-pretreated monocytes, and dexamethasone-pretreated CD4+ plus untreated monocytes. In Fig. 4Go, the highest expression of RANTES mRNA was found in untreated CD4 plus monocytes stimulated with PHA overnight (lane 6; control, lane 5). When the monocytes were pretreated with dexamethasone and added to CD4 cells exposed to PHA (lane 8), the expression of RANTES mRNA was not significantly different from that of the controls (lane 7). Steady state levels of RANTES mRNA on dexamethasone pretreated CD4 cells plus untreated monocytes activated with PHA was detected at 18 hr (lane 10; control, lane 9), but was less than PHA-stimulated untreated CD4 cells plus monocytes.



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  		Fig. 4. RT-PCR analysis. Steady-state levels of RANTES mRNA in CD4 cells (lane 1 without PHA; lane 2 with PHA); monocytes (Mø) (lane 3 without PHA; lane 4 with PHA); CD4 cells + Mø (lane 5 without PHA; lane 6 with PHA); CD4 cells + Mø pretreated with DXM overnight (lane 7 without PHA; lane 8 with PHA); and CD4 cells pretreated with DXM overnight plus monocytes (lane 9 without PHA; lane 10 with PHA). The cells were cultured for 18 hr and stimulated, or not, with PHA (20 µg/ml). The overnight incubation with DXM (10-7M) was approximately 18 hr. Representative results of four independent experiments with cells from four different donors.

 
RANTES production by CD4-primed monocytes activated by PHA.  After isolation of CD4 cells by immunomagnetic separation, the cells were incubated overnight (18 hr) with or without autologous monocytes. Fig. 5Go shows that CD4-primed monocytes, when stimulated with PHA, produce more RANTES than unprimed CD4 cells alone. In addition, the quantity of RANTES secreted by PHA-activated monocytes was less than that produced by CD4 plus monocytes or CD4-primed monocytes. In all the experiments, unprimed CD4 cells activated with PHA generated low quantities of RANTES, which was not different from the quantities generated by PHA-untreated cells. Macrophage-primed CD4 produced much more RANTES (approximately 4 times) than CD4-unprimed cells when they were stimulated with PHA.



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  		Fig. 5. Generation of RANTES by CD4 cells, CD4 cells primed monocytes (Mø), Mø, Mø-primed CD4 cells, and CD4 cells plus Mø, before (control) or after stimulation with PHA (20 µg/ml). The cells were primed for approximately 18 hr. CD4 cells or macrophages were first purified and then put in culture together for 8 hr for priming; they were separated again through adhesion and stimulated, or not, with PHA. Error bars show the SD from quadruplicate experiments (p < 0.005). C = control (untreated cells).

 
Generation of RANTES by CD4+, CD8 and peripheral blood mononuclear cells activated with PHA.  In order to see if CD8 cells or PBMC in toto generate RANTES, we cultured purified CD4+, CD4+ plus CD8+, and PBMC for 18 hr with or without PHA. In these experiments (Fig. 6Go), CD4+ plus CD8+ activated with PHA produced large amounts of RANTES compared to the control (unstimulated cells) or CD4 alone, while PHA-activated PBMC produced a higher quantity of RANTES compared to activated CD4 + CD8+ cells. These results demonstrate that CD4+ plus CD8+ activated cells produce greater quantities of RANTES than activated CD4 alone.



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  		Fig. 6. RANTES production by CD4 cells, CD4 cells plus CD8 cells, and PBMC, before and after stimulation with PHA (20 µg/ml) and incubation for 18 hr. The p values were obtained comparing control (C) with PHA treatments in quadruplicate experiments (NS, not significant).

 
Determination of RANTES mRNA levels on CD4, CD4 plus CD8 and PBMC activated by PHA.  Fig. 7Go shows the levels of RANTES mRNA observed in CD4+, CD4+ plus CD8+, and PBMC activated with PHA and cultured for 18 hr. The level of RANTES mRNA from activated or unactivated CD4 cells was undetectable (lanes 1,2); likewise with inactivated CD4+ plus CD8+ cells (lane 3). When CD4+ plus CD8+ cells were stimulated with PHA, high levels of RANTES mRNA were found (lane 4). However, PHA-activated PBMC (lane 6) produced higher quantities of RANTES compared to the control (lane 5) or CD4+ plus CD8+ activated cells (lane 4).



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  		Fig. 7. RT-PCR analysis. Steady-state levels of RANTES mRNA in CD4 cells (lane 1 without PHA; lane 2 with PHA); CD4 cells plus CD8 cells (lane 3 without PHA; lane 4 with PHA); and PBMC (lane 5 without PHA; lane 6 with PHA); PHA was used at 20 µg/ml and incubations were 18 hr. Representative results of four independent experiments with cells from four different donors.

 
Production of RANTES by CD4 cells and/or monocytes by TNF{alpha} or IL-1.  To determine the generation of RANTES in CD4+ cells and/or monocytes in overnight cell culture using TNF{alpha}, a physiological inducer of RANTES, total peripheral blood mono-nuclear cells (PBMC) and monocytes, alone or in combination with CD4+ cells, were stimulated with TNF{alpha} (10 ng/ml) for 18 hr. Fig. 8Go shows that TNF{alpha} induces significant release of RANTES in monocytes, but not in CD4+ cells. Highly purified CD4 cells plus monocytes produced high levels of RANTES after the exposure to TNF{alpha}. When monocytes were pretreated with dexamethasone (for 18 hr) plus untreated CD4 cells and then activated with TNF{alpha}, the release of RANTES was not significant compared to the controls (unstimulated cells). Dexamethasone-pretreated CD4 cells plus purified monocytes produced large amounts of RANTES, but less than TNF{alpha} activated CD4 cells plus monocytes. TNF{alpha} activated cells paralleled the PHA-stimulated cell data.



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  		Fig. 8. Generation of RANTES by CD4 cells, monocytes (MO), CD4 cells plus MO, CD4 cells plus MO pretreated overnight with dexamethasone, CD4 cells pretreated overnight with dexamethasone plus MO, and PBMC before and after activation with TNF{alpha} (10 ng/ml) incubated for 18 hr. The p values were obtained by comparing control (C) with PHA treatments. Error bars show the SD from quadruplicate experiments (p < 0.005).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Human RANTES tends to attract CD4+ T cells with some preference for memory T lymphocytes with a phenotype CD45RO (UCHL1+ CD4 helper cells associated with memory T-cell functions) [19]. In our studies the secretion of RANTES by human peripheral blood CD4+ lymphocytes, after stimulation with PHA, showed CD4+ cells together with autologous monocytes were the dominant source of RANTES, suggesting a possible effect of cell-to-cell interaction in the induction of RANTES.

RANTES is differentially expressed in CD4+ cell plus monocytes, vs dexamethasone pretreated CD4+ cells plus normal monocytes. CD4+ cells produce more RANTES in combination with monocytes, which would be responsible for the recruitment of more APCs and T cells to the site of inflammation [20,21]. It is not clear if the production of RANTES by CD4+ cells is due to cellular contact with macrophages, or due to activated macrophages releasing a protein, or proteins, that stimulate(s) RANTES secretion. However, in some experiments where macrophages were pretreated with dexamethasone, RANTES was not secreted by CD4 cells. This may indicate that RANTES production from CD4 cells depends on a macrophage product(s), an hypothesis that remains to be tested.

When tumor necrosis factor (TNF-{alpha}) was used as a cell activator for RANTES production [17,22,23], the results showed the same dependance on monocytes. The data indicate that human CD4 cells in combination with monocytes have a unique capacity to secrete RANTES. It is likely that, given their different effector functions, CD4+ cells and monocytes are differentially recruited to peripheral sites of inflammation [24,25]. Here, we report that human CD4+ cells plus untreated monocytes, and CD4+ cells pretreated with dexa-methasone plus unstimulated monocytes, differentially express theß chemokine RANTES, and, accordingly, may differentially migrate in response to different RANTES concentrations. Regarding the very low release of RANTES by CD4+ cells, our results are in accordance with other studies where RANTES is primarily the product of CD8+, rather than CD4+, lymphocytes [26,27].

It appears that monocytes are notably strong drivers of CD4+ cell induction and probably their effector functions, as demonstrated by the enhanced production of RANTES. The mechanism of this macrophage effect on CD4+ cells is unclear. What monocyte product(s) activates CD4+ cells to produce RANTES is not known. Is it a specific cytokine? Or are all macrophage products necessary for this process [28,29]? Ongoing studies in our laboratory are focused on finding the specific product(s) involved in CD4 activation.

Corticosteroids are used therapeutically as potent immunosuppressive and anti-inflammatory agents for a broad spectrum of diseases, including autoimmune and allergic inflammatory diseases and organ transplant rejection [30]. The effectiveness of corticosteroids in these diseases is thought to be due to their capacity to modulate cytokine production in leukocytes and to alter the trafficking and function of neutrophils, lymphocytes, monocytes, etc. [31,32]. Here we found that dexamethasone, besides inhibiting the synthesis of inflammatory cytokines, inhibits RANTES in CD4+ cells and monocytes. This inhibition can lead to a reduced leukocyte infiltration in inflamed sites. Experiments were performed with PHA as a stimulator, since it was previously reported that PHA-stimulated human peripheral blood leukocytes express chemokine protein and mRNA in a dose-dependent manner, while LPS is less effective and, when used in combination with PHA. provokes inhibition of chemokine production by monocytes compared with PHA alone [16].

In summary, although PHA-stimulated CD4+ cells plus autologous monocytes [33] express mRNA and RANTES protein, these effects were not significant in untreated CD4+ cells plus dexamethasone pretreated monocytes. Our observations suggest that the different production of RANTES by CD4+ cells and monocytes in diverse conditions contribute to an understanding of the mechanism by which RANTES profoundly affects inflammatory responses in vivo, and suggests that RANTES may have inhibitory biological effects on inflammatory conditions [32,34,35]. These results underscore the importance of the simultaneous presence of monocytes and CD4+ cells for the production of RANTES in the inflammatory process.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This study was supported by grant 98' (60%) from the Ministry of University, Scientific and Technological Research, Italy. The authors thank Umberto Mortari, Luigi Carratelli, Giuliano De Marco, and Ann Wilkins from Merck, Sharp & Dohme (Italy) for providing some of the laboratory supplies for this study.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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