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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2905-2913
Macrophage Inflammatory Protein-1 Induces Migration and
Activation of Human Thymocytes
By
Daniel J. Dairaghi,
Karin Franz-Bacon,
Eleni Callas,
James Cupp,
Thomas J. Schall,
Susan A. Tamraz,
Stefen A. Boehme,
Naomi Taylor, and
Kevin B. Bacon
From the Departments of Immunology and Molecular Biology, DNAX
Research Institute, Palo Alto, CA; the Department of Immunology,
Neurocrine Biosciences Inc, San Diego, CA; and the Institut de
Genetique Moleculaire, Montpellier, France.
 |
ABSTRACT |
The CC chemokine macrophage inflammatory protein 1 (MIP-1),
has been shown to be a chemoattractant preferentially activating CD4+ CD45RA+ T lymphocytes. Further
analysis of chemokine action on lymphocytic cells has shown the potent
migration-promoting capacity of MIP-1 on human thymocytes. The
responding cells were the CD4+ and CD8+
single-positive (SP), as well as the CD4+
CD8+ double-positive (DP) populations, with little if any
migratory activity on the double-negative (DN) population. The
activation of thymocytes by MIP-1 appeared to be a direct,
receptor-mediated event as evidenced by the rapid mobilization of
intracellular calcium, increase in proteins phosphorylated on tyrosine,
and activation of the mitogen-activated protein kinase (MAPK) pathway. Radioligand binding analyses showed specific and displaceable binding
of MIP-1 to thymocytes with a Kd of approximately 1 nmol/L, a
profile that was comparable with MIP-1 binding to CCR-5-transfected NIH 3T3 cells. In addition, CCR-5 mRNA was detected in total thymocyte populations indicating that activation of thymocytes by MIP-1 may
occur through binding to CCR-5. Further dissection of the subpopulations showed that only the DP and CD8+ SP
populations expressed CCR-5 and expression data on these two populations was confirmed using anti-CCR-5 monoclonal antibody. These
data may be suggestive of a role for MIP-1 in human thymocyte activation, and show a potential route for HIV infectivity in the
developing immune system.
| |
INTRODUCTION |
THE CHEMOKINES ARE a superfamily of small
molecular mass proteins (8 to 16 kD) considered to play an important
role in the induction and maintenance of leukocytic infiltrates in
inflammation.1,2 Presently, the superfamily can be
subdivided into four subfamilies based on the conservation of an
N-terminal cysteine motif. Thus, the CXC subfamily, characterized by
molecules such as IL-8, MGSA/Gro , and NAP-2, are primarily
activators of neutrophils, whereas the CC family, characterized by
among others RANTES, MIP-1, MIP-1, and MCP-1 through 4, are
active on leukocyte populations other than neutrophils.1-4
The CX3C prototype Fractalkine has been shown to
preferentially activate monocytes and T lymphocytes, whereas the
C-family-member lymphotactin thus far preferentially attracts
lymphocytes and natural killer (NK) cells.5-8
We originally hypothesized that chemokines can have leukocyte-specific
chemoattractant effects, after demonstrations of CD4+
CD45RO+ lymphocyte migration induced by RANTES, and
CD8+- and CD4+-specific migration of
lymphocytes induced by MIP-1 and MIP-1, respectively.9-11 However, in heterogenous cell
populations, little is known about the receptors that bind these
chemokines. CC chemokine receptor 1 (CCR-1) shows high affinity binding
for MIP-1 and RANTES.12 Although subsequent cloning
efforts have increased the repertoire of CC chemokine receptors, these
receptors all appear to show a broad specificity of action, binding two
or more of the CC-chemokines.13 Moreover, whereas MIP-1
binds CCR-1 and CCR-5, evidence for specific, competable binding of
MIP-1 leading to signal transduction has thus far only been shown
with transfected CCR-5.14,15
More recently, MIP-1, MIP-1, and RANTES have been shown to act as
soluble inhibitors of infectivity by M-tropic strains of
HIV-1.16 Subsequently, it was shown that CCR-5 acts as an HIV-1 coreceptor mediating infection by M-tropic
strains,17-21 explaining the inhibitory role of these
chemokines. Thus, characterization of cell types expressing functional
CCR-5, or closely related receptors, will be an important exercise in
determining the course of HIV transmission and infectivity.
In our further analyses of the action(s) of MIP-1 on lymphoid cells,
we have found that this chemokine can potently stimulate migration of
human thymocytes. In this report we show that this effect is mediated
by direct receptor ligation, eliciting rapid and transient calcium flux
and tyrosine phosphorylation of multiple protein species. Cell
fractionation has shown that CD4+ CD8+
double-positive (DP) and CD4+ and CD8+
single-positive (SP) thymocytes migrate in response to MIP-. In
addition, thymocytes respond to MIP-1 by activation of
mitogen-activated protein kinase (MAPK). Reverse transcriptase PCR
analyses have also shown the presence of message for CCR-5 in the DP
and CD8+ SP thymocyte subpopulations, and surface
expression was confirmed using anti-CCR-5 monoclonal antibody. Ligand
binding analyses have shown high-affinity binding of this chemokine to
thymocytes, comparable with that detected in CCR-5-transfected NIH 3T3
cells.
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MATERIALS AND METHODS |
Thymocyte preparation.
Human thymus, removed as a consequence of cardiac surgery (average age,
3 months; range, 1 week to 2 years; n = 18), was gently teased apart in
cold Hank's balanced salt solution (HBSS), under sterile conditions.
Total thymocytes were separated after Ficoll density gradient
centrifugation and multiple HBSS washes at 4°C. Minor contaminating
populations of monocytes/macrophages were removed after Dynal bead
separation of anti-CD14-labelled cells according to standard
protocols. Contaminating erythrocytes were rigorously removed by
multiple centrifugation on Ficoll. Populations of whole thymocytes were
then stored on ice before use or stained for fluorescence activated
cell sorting (FACS) of individual subpopulations.
FACS and CCR-5 staining.
For cell sorting, thymocytes were labelled with anti-CD4-fluorescein
isothiocyanate (FITC) and anti-CD8-PE (Becton Dickinson, Mountain
View, CA). After gating on appropriate populations, SP (CD4+ or CD8+) cells, CD4+
CD8+ DP and CD4 CD8 double-negative (DN)
cells were sorted using a FACStar Plus (Becton Dickinson) cell sorter
by standard methods. Sorted populations were used immediately for
bioassay, or frozen at  80°C before RNA preparation.
Surface expression of CCR-5 was assessesed using a monoclonal
anti-CCR-5-FITC antibody according to the manufacturer's instructions (R&D Systems, Minneapolis, MN; IgG2b). Briefly, human thymocytes were
prepared and stained with anti-CD4-PE, anti-CD8-PE-cychrome, and
CCR-5-FITC antibodies as described above. Three color-standard FACS
analyses were performed, with data being collected on 10,000 events,
and CCR-5 expression was assessed on populations gated for
CD4+, CD8+,
CD4/CD8, and
CD4+/CD8+
expression.
Chemotaxis assay.
Chemotaxis of thymocytes was performed by modification of the
previously described method22 by using the 48-well modified Boyden chamber from NeuroProbe (Cabin John, MD). Cells that had migrated onto the underside of an 8 or 5 µm PVPF polycarbonate membrane, after 1-hour incubation at 37°C, were quantified under high power (400x) and results expressed as mean ± SEM for five high-power fields in comparison with lymphotactin-induced migration used as a positive control.
Calcium flux analyses.
Whole thymocyte populations were labelled with indo-1AM (3 µm final
concentration; Molecular Probes, Eugene, OR) for 45 minutes at room temperature. After labelling, cells were resuspended in 1 mL HBSS containing 1% bovine serum albumin (BSA). Measurement of
calcium flux was performed according to previously published methods23 by using a PTI fluorimeter (South Brunswick, NJ).
Phosphorylation analyses.
Unfractionated thymocytes were used for phosphorylation analyses as a
determination of additional signal transduction mechanisms stimulated
by MIP-1. Whole thymocytes were used immediately after separation
from the thymus. Cell viability at the time of assay was consistently
greater than 95%. After agonist stimulation, cell pellets were lysed
in buffer (1% NP-40, 50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl,
0.25% deoxycholate, 5 mmol/L EDTA) containing protease and phosphatase
inhibitors (1 mmol/L PMSF, 10 µg/mL each of aprotinin and leupeptin,
1 mmol/L sodium orthovanadate, 1 mmol/L EGTA, 100 µmol/L
-glycerophosphate, 10 mmol/L sodium fluoride, 1 mmol/L tetrasodium
pyrophosphate). Lysates were mixed with 2x SDS running buffer and
loaded onto 12% tris-glycine gels (Novex; San Diego, CA). Equivalent
loading of samples was determined by BCA assay (Pierce Chemical Co,
Rockford, IL). Separated proteins were transferred to PVDF membranes
(Millipore Corp, Bedford, MA), nonspecific sites blocked with
block-buffer (5% BSA in Tris-buffered saline [TBS], containing 0.1%
[vol/vol] Tween-20 and 0.05% [vol/vol] thimersol) then stained
overnight with antiphosphotyrosine antibody (4G10; UBI). After
staining, phosphorylated proteins were shown with an antimouse IgG-HRP
conjugate, washed three times as above, detected using ECL reagent
(Amersham Corp, Arlington Heights, IL) and Biomax MR film (Eastman
Kodak Co, Rochester, NY).
In parallel, whole cell lysates from thymocytes (5 × 106) stimulated for 3 minutes with MIP-1
(107 mol/L) or 10 µg anti-CD3 (UCHT-1; Sigma) were
run on 10% tris-glycine gels, transferred as detailed above then the
blots stained with anti-ACTIVE MAPK antibody (rabbit polyclonal; 1:5000
dilution; Promega Corp, Madison, WI). Activated MAPK was detected using ECL reagent, as described above. The blot was subsequently stripped and
reprobed with anti-ERK2 (p42 MAPK) antibody (1:1000 dilution; Transduction Laboratories, Lexington, KY).
Reverse transcriptase-polymerase chain reaction (RT-PCR)
analyses of thymocyte populations.
Total thymocytes, or individually-sorted populations (>98% purity; 2 × 106-107 cells) were washed once in
phosphate-buffered saline (PBS) and RNA was extracted using the
S.N.A.P. Total RNA Isolation Kit (Invitrogen, San Diego, CA) according
to the manufacturer's protocol. First-strand cDNA synthesis for use in
PCR reactions was generated using the cDNA Cycle Kit (Invitrogen).
Parallel reactions were set up without AMV RT enzyme in order to
control for genomic DNA contamination in subsequent PCR reactions. PCR
analysis was then performed using the following primers specific for
human CCR-5: sense, 5'-ATGGATTATCAAGTGTCAAGT-CCAATC; antisense,
3'-TCACAAGCCCACAGATATTTCCTGC,14 and 40 cycles of 94°C,
1 minute; 55°C, 2 minutes; 72°C, 3 minutes. Similar reactions were run with human CCR-5 cDNA (positive control), and water in place
of first-strand cDNA (negative control). Amplified product (1.1 Kb) was
resolved on a 1% agarose gel containing ethidium bromide.
Radioligand-binding analyses.
For displacement binding studies, purified human thymocytes were
incubated with 0.2 nmol/L 125 I-labeled chemokine (2200 Ci/mmol/L; DuPont NEN, Boston, MA) for 3 hours at 4°C in the
presence of increasing concentrations of unlabeled chemokine. All
reactions were performed in duplicate and were repeated three to five
times. Each reaction contained 107 cells in a final volume
of 150 µL of binding buffer (PBS, 0.5% BSA, 1 mmol/L
CaCl2, 5 mmol/L MgCl2). The reaction was
terminated by layering the cell suspension over 200 µL oil (80%
silicone oil [Dow Corning 550 fluid] and 20% paraffin oil), and the
cells were separated from the buffer by centrifugation. The tube bottom containing the cell pellet was cut and placed into a fresh vial and the
radioactivity quantified in a Packard Cobra 5010 g counter (Packard
Instruments Co). The binding data was analyzed using Igor Pro software
(Wavemetrics) to determine the displacement binding, Kd,
and the number of binding sites per cell.
| |
RESULTS |
MIP-1 induces chemotaxis of human thymocytes.
Freshly isolated unfractionated thymocytes and sorted DN, DP, CD4+, and
CD8+ SP thymocytes were assayed for chemotaxis in response to MIP-1,
using phytohemagglutinin (PHA) as a control agonist.
Figure 1A shows the mean number of cells migrating in response to PHA (0.01 µg/mL to 100 µg/mL) compared with that
induced by MIP-1. Surprisingly, MIP-1 induced potent migratory
effects on the whole thymocyte population over a broad range of
concentrations ranging from 0.1 nmol/L to 100 nmol/L. Maximal migration
of thymocytes was obtained with 109 mol/L MIP-1 (
Fig 1A).
Further analysis showed that induced migration was occurring within the
DP, CD4+, and C8+ SP populations, but not in
the DN population. The migratory potential of the DN population was
confirmed by their response to lymphotactin (Fig 1B, inset). Little, if
any, difference was noticed in the potency of MIP-1 in stimulating
migration of the individual responder populations (Fig 1B). In all
responder populations, maximal migration was obtained with 1 nmol/L
MIP-1, with significant migration above background occurring with
concentrations as low as 0.01 nmol/L. In contrast, there was
significantly greater migration induced by MIP-1 in the
CD4+ SP and DP populations at subnanomolar concentrations,
with almost double the number of cells responding at 0.1 nmol/L
MIP-1 compared with the CD8+ SP population.
MIP-1 stimulates calcium flux in thymocytes.
Analysis of classical chemokine function prompted an investigation of
the capacity of MIP-1 to stimulate calcium flux in the thymocyte
population. As shown in Fig 2, there was an
elicited flux with unfractionated thymocytes, characterized by a rapid transient flux, in contrast to the more prolonged flux associated with
PHA. These flux profiles are comparable with those obtained using other
chemokines on unfractionated peripheral blood lymphocytes (PBL) [not
shown]. In addition, the short duration of the response was suggestive
of a classical chemokine-chemokine receptor-induced transient
Ca2+ flux.
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| Fig 2.
MIP-1-induced Ca2+ mobilization in
unfractionated human thymocytes. Freshly-isolated cells were loaded
with 3 mmol/L (final) Indo-1AM and kept in a constantly-stirred
cuvette, as detailed in the Materials and Methods section. Stimulation
was with 100 nmol/L recombinant human MIP-1. The inset shows the
flux profile obtained in response to 2 µg/mL PHA as a positive
control. Flux profiles are representative examples taken from n = 6 experiments.
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Phosphorylation analyses.
Analysis of whole cell lysates from freshly-prepared thymocytes
stimulated with MIP-1, showed potent upregulation of tyrosine phosphorylation (Fig 3A). The species
phosphorylated appeared similar to those observed in the
anti-CD3-stimulated thymocytes with one exception; a species of 21 to
24 kD was clearly phosphorylated in the thymocytes in response to
anti-CD3 but not MIP-1. This was shown to be TCR (p24) by Western
analysis (data not shown). Compared with control, unstimulated lysates,
proteins of 120 to 130 kD, 65 to 90 kD, and 40 to 46 kD were
phosphorylated in MIP-1-stimulated cells. Preliminary studies using
monoclonal antibodies in Western analysis of antiphosphotyrosine
immunoprecipitated proteins, have shown that
p130Cas and p56lck constituted
at least two of the phosphorylated species (K.B.B., manuscript in
preparation).
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| Fig 3.
Analysis of tyrosine phosphorylated proteins in
thymocytes in response to MIP-1. (A) Freshly-isolated unfractionated
thymocytes were kept on ice before stimulation wth 10 nmol/L and 100 nmol/L MIP-1, or anti-CD3 as a control, for 3 minutes at 37°C.
After stimulation, the lysates were prepared as described in the
Materials and Methods section. Electrophoresis under reducing
conditions was performed using 12% tris-glycine gels and Western blots
probed with antiphosphotyrosine (clone 4G10). (B) Thymocytes were
purified and stimulated with anti-CD3 (10 mg) or 100 nmol/L MIP-1 as
outlined in the Materials and Methods section. Electrophoresis under
reducing condition was performed using 10% tris-glycine gels and
Western blots probed with antiactive MAPK. Blots were stripped and
reprobed with anti-ERK2 (lower panel) to show equivalent loading.
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The phosphorylation of species of 40 to 46 kD suggested the potential
activation of MAPK. Analyses using antiphosphoMAPK polyclonal antibodies clearly showed activated MAPK from cells treated with either
anti-CD3 or MIP-1 (Fig 3B). No phosphoMAPK was observed with
anti-ERK-1 antibodies (not shown).
Competition-binding analyses.
Binding studies with the unfractionated thymocyte population indicated
displaceable MIP-1 binding, albeit with a unique dissociation profile. Two phases were observed, one consisting of an initial partial
displacement, followed by a second more complete displacement (Fig 4A). Strong initial binding of
125I-MIP-1 was rapidly competed by addition of
unlabelled MIP-1. Addition of this competitor to 1 nmol/L resulted
in a significant reduction in binding, indicating a high-affinity
interaction (Kd of 0.1 nmol/L). For the initial phase,
maximal displacement occured with addition of 1 nmol/L to 5 nmol/L
unlabelled MIP-1. Interestingly, however, addition of MIP-1 to 10 nmol/L resulted in potentiation of total
125I-MIP-1 binding (n = 5) , marking the second phase of
the displacement profile. Addition of unlabelled MIP-1 completely
dissociated the bound 125I-MIP-1. The Kd
value for this latter displacement profile was 35 nmol/L; a value also
derived if all the points are included in the calculations, as shown in
Fig 4A.
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| Fig 4.
(A) Biphasic thymocyte binding of
125I-MIP-1. Initial binding of 125I-MIP-1
to thymocytes is competed by increasing concentrations of unlabelled
MIP-1. Scatchard analysis of all points indicate a total Kd value of
35 nmol/L with an estimated 4,500 sites per thymocyte over the entire
population (not shown). If each phase of the reaction is analyzed
separately, a Kd value of 0.1 nmol/L (first phase; dotted line) and 35 nmol/L (second phase) are determined. This reaction carried out at
4°C contains 70,000 cpm of input 125I-MIP-1 per
reaction, and is representative of n = 5 experiments each using
thymocytes from different donors. (B and C) Heterologous competiton of
125I-MIP-1 bound to thymocytes by MIP-1 and RANTES,
respectively. This result is taken from the same experiment as depicted
in (A). (D) Ligand binding study using 125I-MIP-1 and
CCR-5/NIH 3T3 transfectants. 125I-MIP-1 was added at
120,000 cpm per reaction. (E) and (F) Heterologous competition of
125I-MIP-1 bound to CCR-5/NIH 3T3 cells by MIP-1 and
RANTES, respectively.
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The CC chemokines, MIP-1, and RANTES, which together with MIP-1
are ligands for CCR-5, were able to compete 125I-MIP-1
binding as shown in Fig 4B and 4C, respectively. RANTES and MIP-1
were able to compete the first phase of MIP-1 binding. However, as
also observed with 10 nmol/L MIP-1, after addition of 20 nmol/L
MIP-1 or 500 nmol/L RANTES, the total binding of 125 I-MIP-1 increased significantly. We tested a CCR-5/NIH 3T3
transfectant for comparison with the thymocyte-MIP-1 binding
profile. As shown in Fig 4D, the binding profiles observed with the
thymocytes could be reproduced qualitatively in the transfected cells,
albeit at smaller amplitudes. 125I-MIP-1 binding to the
CCR-5 transfectant could be competed with unlabelled MIP-1, giving
rise to an extended displacement profile. MIP-1 competed
125I-MIP-1 binding with a Ki of 1.0 nmol/L,
whereas RANTES competed with a Ki of 0.3 nmol/L (Fig 4E and
F, respectively). At concentrations of 30 nmol/L MIP-1 or 100 nmol/L
RANTES, there was also a potentiation of 125I-MIP-1
total binding.
Surface expression of CCR-5.
Using a specific antihuman CCR-5 FITC-coupled monoclonal antibody, the
thymocyte population was analyzed for surface expression of CCR-5, the
receptor for which MIP-1 has high affinity. Specific anti-CCR-5
staining was detected on the whole thymocyte population (not shown) so
individual populations were analyzed for surface expression of CCR-5.
Gating on the individual populations, Fig 5
shows that greatest CCR-5 expression was detected on DP populations. There was slight positivity on the CD8+ SP population, however the DN
and CD4 populations were negative.
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| Fig 5.
FACS analyses of CCR-5 surface expression on human
thymocytes. Thymocytes labelled with CD4-PE-cychrome, CD8-PE and
CCR-5-FITC were gated on DN, CD4+, DP, and
CD8+ subpopulations for analyses of CCR-5 expression. The
FACS profiles are representative of experiments using n = 4 different
human thymus donors.
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RT-PCR analyses.
Messenger RNA for CCR-5 was detected using RT-PCR.
Figure 6A shows a representative example of
the expression pattern of CCR-5 mRNA in the unfractionated thymocyte
population, and shows the specificity of the assay, as the message was
absent from the samples lacking reverse transcriptase enzyme. A total
of 2 × 106 thymocytes were used for this experiment,
and the level of specific message corresponds to approximately
102 pg when compared with the CCR-5 cDNA control.
Experiments on sorted populations (Fig 6B) also showed the presence of
CCR-5 in DP and CD8+ SP thymocyte subpopulations,
correlating with the surface expression noted from FACS analyses.
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| Fig 6.
(A) RT-PCR analysis of CCR-5 message in thymocytes.
Messenger RNA for CCR-5 was detected as described in the Materials and Methods section. An unfractionated population of thymocytes (2 × 106 total cells) was used for the reaction. Freshly
isolated thymocytes were analyzed for migration capacity in response to
MIP-1 (not shown) then aliquots frozen for molecular analyses. Human
CCR-5 cDNA, cloned into the pBluescript Sk + vector (Stratagene), was used as a positive control, for the specificity of the primers outlined
in the Methods. (B) Identical experiments were performed with thymocyte
subpopulations (8 × 106) with or without the addition of
reverse transcriptase enzyme.
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CONCLUSION |
Until recently, the biology of MIP-1 has been restricted to limited
reports on the migration- and adhesion-promoting capacity of this CC
chemokine.12,13,24 More recent studies have shown the
ability of MIP-1 to act as a soluble inhibitory factor for HIV entry
into macrophages and T cells.16 Significantly, the receptor
that binds and signals in response to MIP-1, CCR-5, is a specific
coreceptor for macrophage-tropic strains of HIV.17-21 Irrespective of this revelation, little evidence existed for a signal
transducing role of MIP-1 in lymphoid cells, with data only on
activation of T-cell and NK-cell migration. We now show a novel
bioactivity for MIP-1; stimulation of human thymocyte migration in
response to this chemokine. In addition, MIP-1 stimulated intracellular calcium mobilization and induced tyrosine phosphorylation of multiple protein species, including MAPK in thymocytes. Using PCR
primers specific to the CCR-5 gene,14 and a specific
monoclonal antibody to CCR-5 we were also able to detect specific
message and protein expression for this receptor in freshly isolated DP and CD8+ SP thymocytes. This data provides some of the first evidence to support an activation role for this chemokine in lymphoid
populations. The lack of expression of CCR-5 on CD4+ SP
populations does not correlate with the MIP-1-induced migration seen in vitro. Whether or not MIP-1 is activating cell migration through CCR-1, or there is a novel MIP-1-binding receptor on thymocytes, is currently under investigation and such studies will be
enhanced by use of specific neutralizing antibodies in relevant
bioassays.
In equilibrium-binding experiments, MIP-1 showed a unique biphasic
pattern in addition to an increase in 125I-MIP-1
binding. The increase in 125I-MIP-1 binding observed
after addition of 10 nmol/L MIP-1 (or significantly higher
concentrations of MIP-1 and RANTES) is potentially indicative of an
increase in binding sites for this chemokine on thymocytes. This
phenomenon appeared to be unique to 125I-MIP-1 because
neither 125I-MIP-1 nor RANTES gave similar patterns of
binding on thymocytes or the CCR-5 transfectants. The biphasic nature
of 125I-MIP-1 displacement may reflect MIP-1 binding
to two different, but closely related receptors. Because the ligand
binding reactions were performed at 4°C, membrane trafficking
events leading to increased receptor expression can be excluded.
Alternatively, it is possible that CCR-5 or similar receptors undergo
conformational changes in the presence of higher concentrations of
MIP-1 leading to enhanced binding of this specific chemokine with
the receptor. The full molecular characterization of this binding
entity awaits further studies.
Interestingly, as observed in other systems where chemokines stimulated
intracellular calcium mobilization,23,25,26
MIP-1-induced protein tyrosine phosphorylation in lymphoid cells.
The precise role this signaling plays in stimulating thymocytes is
unclear. It is also interesting in so far as PTK activity is only
observed when cells are stimulated immediately after preparation,
having been kept at 4°C. When cells are left at room temperature or
at 37°C, there is a complete loss of apparent PTK signal
transduction capacity. Such a loss of signaling may be the result of
negative regulation of individual signaling components including
kinases known to interact in other chemokine-mediated signal
transduction cascades.27 Further studies are required to
examine such possibilities, as well as the identities of phosphorylated
species of 120 to 130 kD and 69 to 90 kD.
The revelation that p42 MAPK constituted one of the phosphorylated
species of 40 to 46 kD leads us to speculate on the role of this
chemokine in thymocyte activation. This is the first indication of the
induction of MAPK activation in thymocytes using a stimulus other than
anti-CD3. We have not observed proliferative activity in whole
thymocyte populations using MIP-1 as a stimulus in standard assays,
however there may be other subtle effects on specific subpopulations
that are masked by the whole population. It is also interesting that no
obvious activation of p44 MAPK was observed. Potential activation of
p38 MAPK or of alternative JNK/SAPK pathways remains to be assessed to
obtain a more complete picture of MIP-1-induced signal transduction
systems occuring in thymocytes. Interestingly, p38 MAPK phosphorylation
has been detected in IL-8-stimulated human PMN27 and p44
MAPK phosphorylation in MCP-1-activated monocytes,28 both
functions being related to cell migration, thus an investigation of the
relation of this kinase activity to thymocyte migration is a logical
next step.
The biological significance of thymocyte stimulation by MIP-1 is as
yet unclear. Whether this chemokine plays a significant role during
T-cell ontogeny and development, is difficult to determine. The
absolute requirement for IL-7 in T cell commitment29
suggests that MIP-1 may have synergistic and/or additive
signaling roles with other cytokines and chemokines at different stages
of development. The chemokine action may be localized to the
microenvironment of the thymus, recruiting progenitor cells to specific
areas of the thymic superstructure, or retaining them in proximity to
thymic stromal cells. Because the expression pattern of MIP-1 in
pro-T cells and thymocytes shows high frequency,30 this
chemokine may fulfill as yet undescribed functions vital to thymocyte
homeostasis. Interestingly, the DP thymocyte population showed
considerable migratory activity and both the DN and DP subpopulations
exhibit calcium flux responses when stimulated by MIP-1 ( not shown
). Whether or not MIP-1 stimulates the differentiation and
proliferation of these cells into single-positive thymocytes is under
investigation.
One result of major significance is the expression of CCR-5 on
thymocytes. Because CCR-5 is an HIV coreceptor, the presence of this
receptor in the thymic environment potentially provides easy access for
viral infection of the host's developing immune system. Further
studies are now required to extensively catalog the expression pattern
of CCR-5 at different stages of development in neonates. In doing so,
vital information will be obtained concerning the temporal infectivity
pattern of HIV from maternal sources, which may in turn determine
therapeutic regimens with which to combat such infection.
| |
FOOTNOTES |
Submitted August 20, 1997;
accepted November 25, 1997.
The first two authors made equal contributions to this study.
DNAX is supported by Schering Plough Corp.
Address correspondence to Kevin B. Bacon, PhD, Department of
Immunology, Neurocrine Biosciences Inc, 3050 Science Park Rd, San
Diego, CA 92121.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract
