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Pulmonary Center, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of CD4 to function as a coreceptor is not limited to the chemokine receptor family. CD4’s first described role was as a coreceptor for the TCR complex (11, 12, 13). Similar to its relationship with certain chemokine receptors, CD4 directly complexes with the TCR, in the context of Ag and MHC class II binding, and functions to regulate TCR signaling. Normal CD4/TCR/MHC complex formation results in augmented TCR signaling (14, 15, 16), while independent ligation of CD4 by Ab or gp120 results in the inhibition of TCR signaling and induced cell activation (11, 12, 17, 18, 19, 20).
A natural ligand for CD4, IL-16, has been identified and characterized (21) as having a variety of biological effects similar to gp120 stimulation. IL-16 induces CD4+ cell migration (22, 23), alters adhesion molecule expression (24), and facilitates G0 to G1 cell cycle transition in T cells (25). In addition, IL-16 pretreatment results in inhibition of subsequent Ag or anti-CD3-induced cell activation (26, 27). As many of the functions of gp120 mimic the natural ligand, we investigated whether IL-16 binding to CD4 could alter chemokine-induced signaling.
We determined that IL-16 pretreatment results in a selective and
complete inhibition of CCR5-induced migratory signal, as the effects of
MIP-1ß were ablated. Consistent with a specific effect on CCR5 alone,
there was only partial inhibition of both RANTES and MIP-1
stimulation, while monocyte chemoattractant protein (MCP)-2-induced
chemotaxis, signaled through CCR1, CCR2 and CCR3, was unaffected. These
effects were not due to changes in expression or affinity of CCR5 for
MIP-1ß. Unlike IL-16-dependent CD4-mediated chemotaxis, IL-16-induced
CD4-mediated inhibition of MIP-1ß-CCR5-induced chemotaxis required
the presence and enzymatic activity of the SH1 domain on
p56lck. Pretreatment with MIP-1ß resulted in a
desensitization of IL-16-dependent CD4-induced signaling. These studies
suggest a unique natural relationship between CD4 and CCR5 mediated by
either IL-16 or MIP-1ß binding.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human (rh)MIP-1ß, rhRANTES, rhMIP-1, and MCP-1,
-2, and -4 were purchased from Biosource International (Camarillo, CA).
Recombinant murine MIP-1ß was purchased from R&D Systems
(Minneapolis, MN). Fluorescein-conjugated anti-CCR3 Ab (clone 7B11)
and anti-CCR5 Ab (clone 2D7) were kindly provided by Leukosite,
Inc. (Cambridge, MA). Fluorescein-conjugated anti-CD4 and
anti-CD3 Ab were obtained from PharMingen (San Diego, CA).
Unconjugated anti-IL-16 Ab (clone 14.1) was isolated from hybridoma
supernatants, purified using Protein A affinity chromatography, and
used at a concentration of 5 µg/ml, which is sufficient to neutralize
10-10 M rIL-16-induced migration of human T cells
(28).
Cell culture
Normal human T lymphocytes were isolated from the blood of healthy normal volunteers using Hypaque-Ficoll separation of PBMC, as previously described (37). Preparations were enriched for T lymphocytes by nylon wool adherence. The nonadherent mononuclear cells were >95% T lymphocytes, as determined using flow cytometry to assess CD3+ cells. The cells were cultured in medium 199 supplemented with 25 mM HEPES buffer, 100 U/ml penicillin, 100 µg/ml streptomycin, and containing 10% FBS for 18–24 h before use in the chemotaxis assay.
For the studies using herbimycin A, 2 x 106 cells/ml were incubated with herbimycin A (0.5 pM final concentration) (Cal Biochem, San Diego, CA.) for 18 h before washing with culture media. Following washing, rIL-16 (10-10 M) was added to the cells for 15 min, and the cells were washed again and resuspended at 8–10 x 106 cells/ml for use in the chemotaxis assay.
Murine T cell hybridoma cell lines
All CD4-expressing murine T cell hybridomas were a generous gift of Dr. Steven J. Burakoff (Dana-Farber Cancer Institute, Boston, MA). The cells were generated as previously described (29, 30, 31). Briefly, the L3T4-negative murine T cell hybridoma cell line, By155.16, was infected with the MNC retroviral vector that contains a neomycin resistance gene, a CMV promoter, and the gene for human CD4. Various constructs of CD4 (as previously reported, 30) were used to express either wild-type CD4, point mutations corresponding to a Cys420 to Ser mutation, or a Cys430 to Ser mutation. The MNC-CD4 transfectants were selected and assessed for CD4 surface expression. All cell lines used had comparable expression of CD4, as determined by anti-CD4 Ab binding and FACS analysis. In some studies, cells were infected to express a CD4/p56lck chimeric protein. Chimeric molecules containing the extracellular and transmembrane domains of CD4 directly ligated to different fragments of murine Lck were expressed in the By155.16 cell line (31). Neomycin-resistant clones were screened for surface expression of CD4 by flow cytometric analysis using a FACScan (Becton Dickinson, Mountain View, CA). All cells were grown and maintained in RPMI 1640 medium (Sigma, St. Louis, MO) containing 200 U/ml of penicillin and 200 mcg/ml streptomycin, 2 mM glutamine, 20 mM HEPES (pH 7.4), and 10% FBS.
Lymphocyte chemotaxis assay
In vitro chemotaxis was performed as described previously (22, 23). Isolated T cells were pretreated with rIL-16 (10-10 M) or control media for 0–1 h, depending on the experiment, at 37°C, then washed in media twice before use in the chemotaxis assay. In the specificity experiments, the T cells were incubated under the same conditions with rIL-16 (10-10M) and 10 µg/ml IL-16 mAb (14.1), an amount sufficient to neutralize 10-10 M rIL-16 activity (28). Migration was assessed by a modification of a Boyden chamber technique by using a 48-well microchemotaxis chamber (Neuroprobe, Cabin John, MD). Human T lymphocytes (8–10 x 106/ml) or T cell hybridomas (5 x 106/ml), either with or without pretreatment with IL-16, were loaded into the upper well of the chamber, with 32 µl of various concentrations of chemoattractant or control buffer placed in the lower well. The two wells were separated by a nitrocellulose filter paper with a pore size of 8 µm. Chambers were incubated for 2 h for the human cells or 4 h for the T cell hybridomas, after which the filters were fixed in ethanol, stained with hematoxylin, and dehydrated by sequential washes in ethanol, propanol, and the xylene. Cell migration was quantitated by light microscopy by counting the number of cells that had migrated beyond a depth of 50 µm. All migration is expressed as percentage values of cell migration in control media (designated as 100%), and statistics were calculated by the Student t test. All samples were tested in duplicate, and four high-power fields were examined in each duplicate. Data are the mean value ± the SD of three or more experiments.
FACScan analysis
CCR3 and CCR5 expression was analyzed using fluorescein-conjugated anti-CCR3 (clone 7B11) and anti-CCR5 Abs (clone 2D7), and CD4 expression was determined by fluorescein-conjugated anti-CD4 Ab. Human T cells (1 x 106 cells/ml), pretreated and nonpretreated with IL-16, were washed, resuspended in staining buffer, and incubated with 0.25 µg labeled Ab for 30 min at 4°C. Cells were then washed three times in cold staining buffer, resuspended, and fixed with 10% formalin, and analyzed with a FACScan (Becton Dickinson).
Binding assays
Binding assays were conducted by stimulating 3 x 106 human T cells in 100 µl of culture media with either rIL-16 (10–1000 pg) or cold MIP-1ß (8–1000 pg) for 30 min at room temperature before the addition of labeled MIP-1ß. Radiolabeled I125-MIP-1ß (0.12 nM, 185 kBq) (New England Nuclear, Boston, MA) was added to each sample for 120 min at room temperature. Following 120 min of incubation, the samples were quickly aspirated through GF/C microfiber filters (Whatman, Maidstone, U.K.) using a vacuum harvester. The filters were air-dried and counted in a gamma counter. Nonspecific radioactivity present with 10- to 1000-fold excess of cold MIP-1ß was subtracted from total bound counts for each dose and used to calculate specifically bound counts. Percent inhibition of MIP-1ß binding induced by IL-16 was calculated by subtracting counts from IL-16-treated cells from specific counts, divided by specific binding counts, times 100%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether IL-16 could cross-desensitize chemokine
signaling, human T cells were stimulated with IL-16
(10-10M) for 1 h before the induction of
chemokine-stimulated migration. A panel of chemokines was used to
screen functions of CCR1, CCR2, CCR3, and CCR5 (reviewed in Ref.
32). As IL-16 is a chemoattractant, baseline migration was
established by those cells exposed to IL-16 for 1 h with no
further stimulation by any of the chemokines. As shown in Fig. 1
, IL-16 stimulation resulted in the
inhibition of migration induced by RANTES, MIP-1, and MIP-1ß to
varying degrees, while MCP-1, MCP-2, and MCP-4-induced migration was
not affected. Migration induced by MIP-1ß was completely blocked, as
compared with only partial inhibition for migration induced by either
MIP-1 or RANTES (Fig. 1). RANTES utilizes CCR1, CCR3, and CCR5 for
binding; MIP-1 binds to CCR1 and CCR5; MCP-1 uses CCR2; MCP-2 binds
to CCR1, CCR2, and CCR3; MCP-4 binds to CCR2 and CCR3; while the only
known receptor for MIP-1ß is CCR5. Taken together, these data suggest
that IL-16 pretreatment selectively inhibits CCR5-induced migration
(32). Based on this specificity of IL-16 to alter CCR5
stimulation, all subsequent studies focus primarily on MIP-1ß
stimulation, with MCP-2 used as a chemokine "negative" control.
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). However, IL-16 pretreatment of 10 min
or longer did result in complete blocking (Fig. 2). When IL-16 was
combined with a neutralizing concentration of anti-IL-16 Ab 10–15
min before the addition to the cells, the inhibitory effect of IL-16
was negated (data not shown). The relatively short time requirement of
10 min to induce an IL-16-inhibitory effect on MIP-1ß signaling
suggested that the mechanism of blocking was not associated with CCR5
modulation from the cell surface.
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A; only the 24 h time
point is shown). In addition, there was no inhibition of radiolabeled
I125-MIP-1ß binding to human T cells treated
with IL-16 (10-10 M) for 30 min. (Fig. 3B). Thus, desensitization induced by IL-16/CD4 is not
related to surface modulation of CCR5, nor to a reduction in binding
affinity. To control for steric inhibition by IL-16, MIP-1ß binding
was conducted on cells that were not washed following IL-16 treatment.
The ability of MIP-1ß to bind to these cells was identical to that
seen when using cells that were washed following IL-16 exposure (data
not shown), suggesting that there is no direct competition for binding
between IL-16 and MIP-1ß.
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We next investigated whether there was a reciprocal effect
mediated by MIP-1ß-CCR5 stimulation on IL-16-CD4-induced chemotaxis.
Human T cells were stimulated with either MIP-1ß or MCP-2 for 1
h, and the cells were washed and then stimulated by IL-16, in a
dose-dependent fashion, for the induction of chemotaxis. As shown in
Fig. 4, MIP-1ß stimulation did result
in a complete loss of IL-16-induced chemoattraction. MCP-2 stimulation
had no effect on IL-16-induced migration (Fig. 4). A time course was
established to determine the minimal time requirement for MIP-1ß to
desensitize CD4 signaling. Human T cells were stimulated with MIP-1ß
for 5, 10, 20, 30, or 60 min before washing and assessing for IL-16
responsiveness. As shown in Fig. 5,
MIP-1ß stimulation required at least 20 min of stimulation to
significantly effect IL-16-induced migration.
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The observation that IL-16 stimulation did not result in loss of
CCR5 expression from the cell surface or a decrease in MIP-1ß binding
suggested that IL-16/CD4 association resulted in inhibition of an
essential signal. To further define the mechanism by which IL-16
inhibited MIP-1ß signaling, murine T cell hybridomas were transfected
to express point-mutated human CD4 molecules. L3T4-negative T cells
were transfected to express either wild-type CD4; a
Cys420 to Ser (CS420); or
Cys430 to Ser (CS430)
point-mutated CD4 molecules. The CS420 mutation
results in disruption of a CD4/p56lck
association, while mutation of CS430 does not
alter this association (29, 30). These cells have
previously been used to demonstrate that a
CD4/p56lck association is required to confer
IL-16-induced cell migration and second messenger signaling
(31). To determine the effects of IL-16 on CCR5-induced
migration, it was first established that murine MIP-1ß and human
MCP-2 could induce a migratory response in these cell lines similar to
what was observed for human T cells. As shown in Fig. 6, both MIP-1ß and MCP-2 induced
migratory responses, in a dose-dependent fashion. Cells expressing
wild-type CD4, CS420, or
CS430 were then exposed to IL-16 for 1 h and
stimulated with the two chemokines. IL-16, as expected, blocked
MIP-1ß, but not MCP-2-induced migration, in cells expressing the
wild-type or CS430 CD4 molecules; however, there
was no IL-16 effect in the CS420-expressing cells
(Fig. 6). This finding was consistent with the requirement for a
CD4/p56lck association for a direct CD4-induced
migratory signal.
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The IL-16 migratory signal initiated through CD4 is transduced by
an interaction of PI3 kinase with the SH3 domain on
p56lck (31). In addition,
IL-16-induced migration is insensitive to the Src tyrosine kinase
inhibitor, herbimycin A, indicating that, while a physical association
of CD4 with p56lck is required, enzymatic
activity of p56lck is not required for this
signaling. To determine whether the inhibitory effects of IL-16
initiated through interaction with CD4 were also independent of
p56lck enzymatic activity, we incubated isolated
human T cells with the specific Src tyrosine kinase inhibitor
herbimycin A. Cell migration induced by CCR5 has previously been shown
to be herbimycin A-insensitive (33). As shown in Fig. 7, cells treated with herbimycin A, in
addition to pretreatment with IL-16, demonstrated normal responses to
MIP-1ß stimulation. Thus, treatment with herbimycin A blocked the
inhibitory effects of IL-16 stimulation, suggesting that signaling
through CD4 required the enzymatic activity of
p56lck. The inhibitory effect of herbimycin A is
not restricted to the activity of p56lck;
therefore, to confirm these findings, murine hybridoma T cells were
transfected with several different CD4/p56lck
chimeric constructs. These cells, as previously described
(31), express chimeric constructs of wild-type CD4 with
either wild-type p56lck or a deletional mutation
of p56lck, which lacks the SH1 (enzymatic)
domain, synthesized on the C-terminal end of CD4. Cells expressing both
of these constructs have previously been shown to be responsive to
IL-16 stimulation (31). For these studies, an initial
dose-response curve demonstrated that each of the these cell types was
responsive to either MIP-1ß (Fig. 8A) or MCP-2 (data not shown)
stimulation. The same dose response was repeated following a 1-h
incubation in the presence of IL-16 (10-10 M).
Similar to our findings with primary T cells, hybridoma cells
expressing the wild-type p56lck construct,
exposed to IL-16, were subsequently not responsive to MIP-1ß
stimulation (Fig. 8A). Conversely, cells expressing the SH1
deletional mutation did not demonstrate an IL-16-induced inhibitory
effect and responded normally to MIP-1ß stimulation (Fig. 8A). These data indicate that both the presence of the SH1
domain and its enzymatic activity are required to transmit an
IL-16/CD4-mediated signal that blocks CCR5 signaling.
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B, MIP-1ß stimulation had the same effect on cells
either expressing the full-length CD4/p56lck
construct or the SH1 deletional mutation, indicating that the SH1
domain is not reciprocally involved in CD4-initiated CCR5
cross-desensitization. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While all chemokine receptors are seven membrane-spanning proteins, cross-desensitization does not occur between all chemokine receptors, indication of a selective process. As mentioned, this process is also not restricted to just seven membrane-spanning receptors (35). CD4, a single membrane-spanning receptor, is classified as a coreceptor for the TCR (11, 12, 13), a seven membrane-spanning receptor complex. The first identified function for CD4, following Ab binding, was inhibition of Ag or anti-CD3-induced cell activation via TCR stimulation (11, 12). Therefore, stimulation by a CD4 ligand before TCR stimulation results in deactivation of the TCR signaling complex. This effect likely occurs as a result of two independent mechanisms. The first mechanism involves the recruitment of CD4 to complex with the TCR in conjunction with MHC class II, induced by Ag activation. CD4 ligation may therefore prevent CD4’s association with the complex, reducing TCR signaling efficiency. The second mechanism relates to the ability of a CD4/p56lck association to phosphorylate a signaling component of TCR, ZAP 70, thus preventing normal ZAP 70 signal transduction. Utilization of both of these mechanisms would explain why a variety of signaling (HIV-1 gp120 and IL-16) as well as nonsignaling (uncross-linked anti-CD4 Ab) CD4 ligands have been shown to block Ag activation.
In this respect, CD4 and certain of the chemokine receptors can also function as coreceptors. They are co-utilized by HIV-1 virus for cellular entry (3, 4). HIV-1 envelop glycoprotein, gp120, andseveral chemokines recognize overlapping epitopes, and HIV infection is blocked by the presence of certain chemokines or chemokine-derived antagonists (2, 3, 4, 5). As a coreceptor relationship has been established for CD4 and some of the chemokine receptors in the context of gp120 binding, we investigated whether a natural ligand for CD4, IL-16, could also induce a similar relationship.
In these studies, we demonstrate that IL-16, interacting through CD4,
transduces a signal that completely blocks MIP-1ß-induced migration.
The inhibitory effects were only partial for MIP-1 and RANTES
stimulation, while IL-16 had no effect on MCP-1, MCP-2, or MCP-4
stimulation. These data indicate first that the effects of IL-16 are
selective and not as a result of general blocking of all chemokine
signaling. Second, based on multiple receptor usage for many of the
chemokines, our data indicate that the inhibitory effects of CD4
signaling are specific for at least CCR5-induced migration. Based on
the lack of inhibition of MCP-1 stimulation, it appears that signaling
through CCR2 is not effected. Studies using eotaxin are ongoing to
determine the specific effect on CCR3 signaling; however, since
stimulation by MCP-4 was also not altered, it would appear thus far
that desensitization by IL-16 is restricted to CCR5. In addition,
implied by the data is that the migratory signal induced by RANTES and
MIP-1 is the result of signaling through the combination of CCR5 as
well as CCR1 or CCR1 and CCR3 for MIP-1 and RANTES,
respectively.
The mechanism for blocking MIP-1ß, induced by an IL-16/CD4 interaction, does not involve modulation of CCR5 from the surface, nor altering the binding affinity of MIP-1ß for CCR5, as neither of these bioactivities was affected by IL-16 stimulation. These findings suggest that receptor cross-desensitization is induced by generation of a regulatory signal or depletion of a common pathway component. Any regulatory signal would appear to require the presence and enzymatic activity of the CD4-associated Src tyrosine kinase p56lck, as the inhibition was herbimycin A-sensitive and, more importantly, the response was completely lost in cells expressing an SH1 deletional mutation. Blocking of MIP-1ß-induced migration required stimulation of IL-16 for at least 10 min. This time course is consistent with the time frame of p56lck phosphorylation induced by IL-16 stimulation (31). Interestingly, there was a reciprocal blocking effect on IL-16-induced migration when the cells were prestimulated with MIP-1ß. This effect, however, was independent of p56lck involvement since the SH1 deletional mutated cells demonstrated complete blocking of the IL-16 chemotactic response by MIP-1ß. Whether or not other domains on p56lck are required for CCR5-induced inhibition cannot be determined as the SH3 domain is essential for an IL-16-mediated chemoattractant response.
IL-16 and HIV-1 gp120 both bind to CD4, and both have been shown to block chemokine signaling; however, the mechanisms appear to be quite different. In the case of gp120, T cells stimulated with gp120 demonstrate a rapid and almost complete loss of CD4 expression on the cell membrane (19). While the effect of gp120 stimulation on chemokine receptors in lymphocytes has not been reported, the loss of CD4 in monocytes is accompanied by a loss of binding sites for a variety of chemokines in addition to internalization of at least one chemokine receptor, CXCR4, from the cell surface (10). Interestingly, the loss of chemokine binding is not restricted to CD4-associated chemokine receptors, as binding by MCP-2 and MCP-1 is lost. In addition, signaling by fMLP in monocytes is also inhibited, suggesting that gp120 stimulation of monocytes results in a general suppressive condition whereby the cells are unresponsive to all stimulation.
In regards to IL-16, which also binds to CD4, the binding site is different from the epitope for gp120 (26). HIV-1 gp120 binds in the D1-D2 region of CD4, while IL-16 binding has been localized to the D4 region (36). It is not clear at present whether a different binding epitope for IL-16 accounts for the functional differences; however, IL-16 stimulation does not induce modulation of CD4 from the cell surface of T cells (26), nor does it affect surface expression of either CCR5 or CCR3. The inhibitory effect of IL-16 appears to be restricted to CCR5 signaling, as chemotaxis mediated by CCR1, CCR2, or CCR3, induced by MCP-2, was not altered. Since MIP-1ß, but not MCP-2, cross-desensitized IL-16/CD4 signaling, it is possible that a natural coreceptor relationship exists between CD4 and CCR5, induced by either cytokine, which is functionally different from the relationship induced by gp120 binding.
Along those lines, gp120 binding to CD4 induces a conformational change in gp120 that allows for its interaction with the coreceptor CCR5. This concept is supported by the observations that binding of gp120 blocks subsequent MIP-1ß binding (8, 9). In addition, recent studies have identified that, while gp120 stimulation can induce CD4-dependent signaling (migration, p56lck phosphorylation), signaling and cellular responses induced by gp120 can also be transmitted by CCR5 coreceptor association (migration, calcium) (7). These findings were confirmed by studies indicating G protein involvement in gp120 signaling (7).
It is conceivable, therefore, that the inhibitory activity of IL-16 on
CCR5 signaling is as a result of a coreceptor relationship by IL-16 for
both CD4 and CCR5. This would imply that a component of IL-16-induced
migration involves some signaling interaction with a G-coupled protein.
Of interest, IL-16-induced chemotaxis is partially inhibited by
pertussis toxin (our unpublished observations), and there is reported
GTP-binding, Gi, GTPase activity associated with CD4
(37). However, the functional relationship of the G
protein with CD4 signaling is unclear. If a direct interaction of IL-16
with a chemokine receptor (CCR5) does exist, it could contribute to a
CD4-induced migratory signal as well as the reported calcium flux.
While signaling through a chemokine coreceptor could account for many
of the observed bioactivities induced by an IL-16/CD4 interaction, not
all functions can be taken into account. The ability of IL-16 to
inhibit a mixed lymphocyte reaction is in clear distinction with the
effects of many of the chemokines, including MIP-1ß, that have been
shown to augment Ag activation (38). Therefore, without
direct evidence for a CD4-CCR5 coreceptor relationship for IL-16 (i.e.,
induced complex formation following IL-16 stimulation), the
relationship remains unclear.
We can only speculate at present as to the in vivo relevance of these findings. A variety of mechanisms have been hypothesized to facilitate selectivity of recruited cells to sites of inflammation. As IL-16 and MIP-1ß are both generated and detected at sites of inflammation, the opportunity exists for reciprocal cross-regulation of recruited cells.
In summary, our findings suggest that there is a functional relationship between two chemoattractant cytokines of completely different classes, IL-16 and MIP-1ß. A regulatory component exists, whereby pretreatment with one of the cytokines results in cross-desensitization of the other. The mechanism for the IL-16 effect appears to be quite distinct from the mechanism used by HIV-1 gp120, as inhibition occurs as a result of a p56lck-dependent inhibitory signal alone rather than by modulating MIP-1ß binding sites. As both of these cytokines have been detected coincidentally in association with inflammation, it is feasible to hypothesize that this represents an adaptive process to restrict and regulate recruitment of immune cells to sites of inflammation.
Note added in Proof.
Xiao et al. have recently reported on the constitutive cell surface expression between CD4 and CCR5 in T cell lines as well as primary T cells. (Proc. Natl. Acad. Sci. USA 96:7496).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William Cruikshank, Pulmonary Center, R-304, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118. E-mail address:
3 Abbreviations used in this paper: MIP-1ß, macrophage-inflammatory protein-1ß; MCP, monocyte chemoattractant protein; rh, recombinant human.
Received for publication April 14, 1999. Accepted for publication July 6, 1999.
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J. Masters, A. A. Hinek, S. Uddin, L. C. Platanias, W. Zeng, G. McFadden, and E. N. Fish Poxvirus Infection Rapidly Activates Tyrosine Kinase Signal Transduction J. Biol. Chem., December 14, 2001; 276(51): 48371 - 48375. [Abstract] [Full Text] [PDF]
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F. F. Little, W. W. Cruikshank, and D. M. Center IL-9 Stimulates Release of Chemotactic Factors from Human Bronchial Epithelial Cells Am. J. Respir. Cell Mol. Biol., September 1, 2001; 25(3): 347 - 352. [Abstract] [Full Text] [PDF]
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C. Van Drenth, A. Jenkins, L. Ledwich, T. C. Ryan, M. V. Mashikian, W. Brazer, D. M. Center, and W. W. Cruikshank Desensitization of CXC Chemokine Receptor 4, Mediated by IL-16/CD4, Is Independent of p56lck Enzymatic Activity J. Immunol., December 1, 2000; 165(11): 6356 - 6363. [Abstract] [Full Text] [PDF]
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) or IL-16 (•) for 30 min before the
addition of radiolabeled MIP-1ß. The doses of cold MIP-1ß and IL-16
effected up to a 1000-fold excess, as compared with the amount of
labeled MIP-1ß. Cells were incubated for 120 min before filtration
through microfiber filters and assessment of binding using a gamma
counter. Percent inhibition was calculated by subtracting counts from
IL-16-treated cells from specifically bound counts, divided by
specifically bound counts, times 100%.
