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*
Department of Physiology and Biophysics, and
The Price Institute of Surgical Research, Department of Surgery, University of Louisville, School of Medicine, Louisville, KY 40292; and
Health Care Discovery, Novo Nordisk, Novo Allé, Bagsvaerd, Denmark
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, and IL-6) from isolated human monocytes. To date, the
mechanisms by which HBP enhances LPS-induced monocyte activation have
not been elucidated, and it is not known whether HBP also increases the
LPS-induced production of other bioactive substances. We studied human
monocytes activated by recombinant human HBP and LPS and their
interaction with the LPS receptor CD14. We hypothesized that the
stimulatory effect of HBP on the LPS-induced release of proinflammatory
mediators from monocytes was mediated by specific binding of HBP to
monocytes, which resulted in an up-regulation of CD14. Our results
demonstrated that HBP alone (10 µg/ml) stimulated the production of
TNF- from isolated monocytes. In addition, HBP had an additive
effect on LPS-induced production of TNF- and PGE2,
suggesting a generalized monocyte activation. We used flow cytometry to
demonstrate that HBP had a high affinity to monocytes but not to the
LPS receptor CD14, and experiments performed at 4°C indicated an
energy-dependent step in this process. Confocal microscopy showed that
monocytes internalize HBP within 30 min. These data suggest that
mechanisms other than increased CD14 expression are responsible for the
enhanced release of TNF- or PGE2 in response to HBP and
LPS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-6, and therefore modulate both T and B
cell function. Another important role of neutrophils in the late stages
of infection is the release of "classical" chemokines such as IL-8
(3) or other multifunctional proteins from azurophilic granules. There
is increasing evidence that one such protein is a cationic
antimicrobial protein with a molecular mass of 37 kDa (CAP37), which is
also known as azurocidin or heparin-binding protein
(HBP)3 (4). Sequence analysis of HBP (5, 6) indicates that this protein bears many similarities to serine proteases, which are important in inflammatory processes (7, 8). The closest sequence homologies were found with neutrophil elastase (47%) and protease 3 (42%), also known as myeloblastin and p29. To a lesser extent, sequence homologies were found with cathepsin G (37%) and serine proteases from cytolytic T lymphocytes. However, even though HBP is a member of the serine protease family, it lacks protease activity due to mutations of two of the three amino acids in the highly conserved catalytic triad—the histidine and serine residues have been changed to glutamine and tyrosine, respectively (9, 10).
Despite its lack of proteolytic activity, HBP has a variety of
physiologic functions, and when HBP is released from neutrophils, it
has a high potential for regulating monocyte function (4). In contrast
to the intracellular release of many antibiotic proteins such as
defensins or bactericidal permeability-increasing protein (BPI), 89%
of neutrophil-derived HBP is released extracellularly during
phagocytosis of Staphylococcus aureus (7). HBP could
therefore be the molecule responsible for the second wave of
mononuclear cells in certain inflammations (4). HBP is a
multifunctional protein with specific and powerful chemotactic
properties for monocytes (7). Furthermore, HBP not only attracts
monocytes but also increases monocyte survival (11), activates
monocytes to secrete thrombospondin (11), and increases LPS-induced
monocyte production of the proinflammatory cytokines TNF-, IL-1, and
IL-6 (12). In addition, HBP has antimicrobial activities at pH values
found in maturing phagosomes (13). The bactericidal site of action
appears to localize to the inner membrane of Gram-negative bacteria,
and initial events involve HBP binding to the lipid A moiety of
LPS (14, 15).
Clearly, the membrane-bound CD14 receptor plays a major role in LPS-mediated monocyte activation (16). However, other LPS receptors such as the ß-2 integrin subunit CD18, which is involved in the nonopsonic recognition of LPS, or an acetyl lower density lipoprotein receptor on monocytes, which is involved in the uptake and detoxification of LPS/lipid A, may also play a role in monocyte activation (17). At least one serum protein, LPS-binding protein (LBP), catalyzes the effects of LPS on monocytes that are mediated through the CD14 receptor (16, 18).
We hypothesized that the stimulatory effects of HBP on the LPS-induced
release of TNF- from monocytes (12) are mediated by specific binding
of HBP to monocytes and up-regulation of membrane-bound CD14. We
demonstrate that HBP alone induces the release of TNF- and that HBP
has an additive effect on LPS-induced release of the proinflammatory
mediators TNF- and PGE2. HBP has a high affinity to
monocytes and is internalized within 30 min. Contrary to our
hypothesis, HBP did not increase CD14 expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HBP was expressed in Spodopterea frugiperda (SF9) cells (Invitrogen, Abingdon, U.K.) and purified as previously described (12). Briefly, we constructed a 770-bp BamHI- HindIII fragment from a human bone marrow cDNA library (Clontech Laboratories, Heidelberg, Germany) using PCR technology. We then inserted the fragment into the baculovirus transfer vector pBlueBacIII (Invitrogen), which resulted in the transfer plasmid pSX556. SF9 cells were transfected using linear Autographica california nuclear polyhedrosis virus DNA and transfection plasmid (Invitrogen). We collected the insect cell culture medium 3 to 4 days after transfection and purified HBP by glass microfiber filtration (GF/A, Whatman, Meadstone, U.K.), CM-Sepharose cation exchange columns, and Sephadex G-25 gel filtration columns (Pharmacia, Zurich, Switzerland).
Reagents and mAbs
Mouse anti-human CD14, FITC, and phycoerythrin (PE)-coupled Mo2 (Coulter, Hialeah, FL) were used to tag monocytes or measure CD14 expression on monocytes. Purified mouse anti-human CD14 (MY4, 20 µg/ml, Coulter, Hialeah, FL) was used to block CD14. Mouse anti-human CD18 (unlabeled and FITC-coupled clone IB4, Ancell, Bayport, MN) was used to block CD18 and to assess CD18 expression on monocytes, respectively. Isotype-matched FITC- and PE-coupled Ig (Becton Dickinson, Rutherford, NJ) were used as a control for CD marker expression. Isotype-matched IgG2 (Ancell) was used as a control in the Ab studies. Heparin was obtained from Elkin-Sinn (Cherry Hill, NJ). Escherichia coli 011:B4 LPS, EDTA, dextran-500, RPMI with glutamine, and HBSS with Ca2+ and Mg2+ were purchased from Sigma Chemical (St. Louis, MO). The Limulus amebocyte lysate assay was purchased from Associates of Cape Cod (Woods Hole, MA). Sterile and endotoxin-free FCS (LPS concentration < 0.05 ng/ml) was purchased from BioWhittaker (Walkersville, MD).
Affinity studies for LPS and HBP
Whole blood was collected from healthy volunteers and stored in acid citrate dextrose vacutainers at room temperature until the studies were conducted. For the LPS binding studies, we used 10 µg/ml FITC-LPS E. coli 055:B5 (Sigma Chemical). To assess the affinity of HBP to leukocytes, we used a concentration of 10 µg/ml FITC-HBP, a dose based on the literature (11) and on preliminary experiments from our laboratory (unpublished data). Initial affinity and time response studies were determined in 12 mm x 75 mm polypropylene tubes with a final volume of 600 µl/tube in a shaking water bath at 37°C.
In subsequent studies, we determined the affinity of FITC-HBP and FITC-LPS in 50 µl of whole blood in microcentrifugation tubes at 37°C with 5% CO2 or at 4°C. Meticulous care for temperature control was necessary in the later experiments. In the affinity studies performed at 4°C, the samples were placed on melting ice and incubated in the refrigerator. Preincubation was performed for 60 min, and coincubation consisted of the concurrent addition of the reagents, unless stated otherwise. At the end of the experiments, erythrocytes were removed by hypotonic lysis, samples were washed twice with FTA-azide (Becton Dickinson, Cockeysville, MD), fixed in 1% paraformaldehyde, and analyzed by flow cytometry.
Monocyte isolation and culture
Human monocyte cells were isolated by dextran sedimentation and density gradient centrifugation (19). Briefly, whole blood was collected in EDTA vacutainers, and one part of 6% dextran-500 in 0.9% saline (w/v) was added to 10 parts of EDTA-blood. Leukocyte-rich plasma was harvested after 45 min of sedimentation and layered on top of 3 ml of 1-Step-Monocyte (1068 gradient; Accurate Scientific, Westbury, NY). The gradient was centrifuged at 600 x g for 15 min at room temperature. The upper layer consisted of plasma and was discarded. The middle layer contained the monocytes and was harvested and washed twice with a washing solution containing 0.9% saline, 0.13% EDTA, and 1% FCS. The cell suspension was centrifuged for 7 min at 600 x g and eventually resuspended in culture medium. Culture medium (RPMI 1640 with glutamine) was supplemented with 1% FCS (BioWhittaker), 1% antibiotics (100 µg/ml streptomycin, 100 U/ml penicillin; BioWhittaker), and 1% antimycotics (0.25 µg/ml, amphotericin B; BioWhittaker). The cells were counted with a hemocytometer, and the percentage of CD14-positive monocytes was assessed by flow cytometry. A total of 2 x 105 cells in 1 ml supplemented culture medium was added to each well (24-well plate from Costar, Cambridge, MA) and incubated at 37°C with 5% CO2. The LPS concentration in the supplemented culture medium was < 0.03 ng/ml, as assessed with the Limulus amebocyte lysate assay.
Flow cytometry
A FACScan emitting an argon laser beam at 488 nm (Becton Dickinson, Immunocytometry Systems, San Jose, CA) was used. Fluorescence values were collected after gating cells based on the combination of forward scatter (FSC) and sideways light scatter (SSC). A total of 5000 cells was analyzed per tube, and acquired data were processed using CellQuest version 1.2 software (Becton Dickinson, Immunocytometry Systems). The fluorescence distributions were displayed as single histograms for fluorescence measured at 530 nm (FL1-H) or fluorescence measured at 580 nm (FL2-H). The percentage of fluorescent cells and the mean fluorescence intensity (MFI) were determined in each case. The signals were acquired in a linear mode for FSC and SSC and in a logarithmic mode for FL1-H and FL2-H. The threshold levels were set according to the negative control. The gates for human monocytes, lymphocytes, and neutrophils were set according to the standard position in the SSC and the FSC (20).
Measurement of proinflammatory mediators
An ELISA was used to determine levels of TNF- (Biosource,
Camarillo, CA) and PGE2 (Cayman Chemical, Ann
Arbor, MI).
Confocal microscopy
Monocytes were isolated as described, transferred to microcentrifugation tubes, and incubated with HBP (10 µg/ml) for 15 min, 30 min, 45 min, 60 min, and 180 min. Samples were transferred to coverglass chambers (Nunc, Naperville, IL), and fluorescence was assessed with confocal microscopy (Meridian, Okemos, MI). In experiments with dual labeling (60 min incubation with FITC-HBP and labeling of monocyte CD14 with Mo2-PE), the calibration and accuracy of the two filters (530 nm for FITC-fluorescence; 580 nm for PE-fluorescence) was tested using anti-CD14 (Mo2-PE), anti-CD18 (IB4-FITC), and a combination of both mAbs. Data were processed with the software provided by the manufacturer (Meridian) and assembled with Photoshop 4.0 (Adobe Systems, San Jose, CA).
Statistical analysis
Statistical significance was determined with ANOVA and Fisher’s probable least-squares difference analysis (Statview 4.5, Abacus Concepts, Berkeley, CA) to compare data between multiple groups at each time period. Student’s t tests was used to compare the data between two groups. A p value of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In initial experiments, we assessed dose responses for LPS and
HBP. Isolated human monocytes were incubated for 24 h with
increasing concentrations of LPS or HBP. TNF- release was measured
by ELISA. The TNF- values for each donor are shown in Figure 1
and demonstrate different individual
sensitivities to LPS (Fig. 1A) and HBP (Fig. 1B). Donor A (solid circles) first responded to 1.25
ng/ml LPS, whereas the other donors needed a LPS concentration four
times higher to release a similar amount of TNF- (Fig. 1A). Donor A also had one of the highest responses to
HBP (Fig. 1B). Donor E had a relatively low response
to LPS and a low-to-moderate response to HBP. Overall, LPS produced an
increase in TNF- release that was significant at LPS concentrations
of 5 ng/ml and reached a plateau between 10 and 20 ng/ml. Because of
the large variability in the individual responses, HBP did not produce
a significant mean increase in TNF- until 10 µg/ml was used.
Differences in individual responses to HBP could not be attributed to
the sex of the subject (data not shown).
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and PGE2 (Fig. 2). The release of TNF- (Fig. 2A) was significantly increased after 10 µg/ml HBP
(gray bar) and 10 ng/ml or 10 µg/ml LPS (hatched bars). The
combination of HBP with either dose of LPS (black bars) significantly
increased the release of TNF- compared with LPS only (hatched bars).
The release of PGE2 was less sensitive to HBP or LPS (Fig. 2B). HBP (gray bar) did not significantly increase
PGE2 production, and only the high concentration of LPS
(hatched bars) induced PGE2 release. However, the
combination of HBP with either dose of LPS (black bars) increased
PGE2 above that obtained with LPS alone (hatched bars).
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production from monocytes. We therefore investigated
the impact of the sequential addition of HBP and LPS on TNF- release
in a separate experiment. Five donors were used in each of three
groups: 1) 30 min of monocyte incubation with 10 µg/ml of HBP, and
then 10 ng/ml of LPS was added for 24 h (TNF-: 2690 ± 411
pg/ml); 2) 30 min of LPS exposure followed by 24 h of HBP exposure
(TNF-: 2454 ± 318 pg/ml); and 3) a group in which HBP and LPS
were premixed for 30 min before the mixture was added to monocytes for
24 h (TNF-: 2454 ± 318 pg/ml). The results were not
statistically different, suggesting that sequentially adding HBP and
LPS to monocytes does not alter the functional effect of HBP on the
enhancement of LPS-induced TNF- production. Binding of HBP to leukocytes
The effects of HBP on the release of TNF- and
PGE2 from monocytes could result from the binding of
HBP to the monocyte. To test this, HBP was conjugated with FITC
(FITC-HBP), and the conjugate was incubated with whole blood for 60 min
at 37°C. HBP-binding to various leukocyte populations was determined
by flow cytometry. Leukocytes were gated into three groups (Fig. 3A) representing
lymphocytes (Fig. 3B), monocytes (Fig. 3C), and granulocytes (Fig. 3D).
FITC-HBP showed a differential pattern of affinity to the leukocytes.
Monocytes had the highest affinity for FITC-HBP, as reflected by a
shift in fluorescence intensity units when compared with control
FITC-IgG (Fig. 3C). Both lymphocytes and granulocytes
showed very little binding of HBP as reflected by the very small shift
in fluorescence compared with that of the control FITC-IgG.
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. At 2
min, the percentage of monocytes with FITC-HBP binding began to
increase. At 10 min, this increase was significantly greater than that
of the lymphocytes or granulocytes, and it peaked at 30 min.
Lymphocytes and granulocytes showed no significant binding to FITC-HBP,
even after 180 min of incubation.
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Role of CD14.
The affinity of HBP to monocytes could be mediated by binding to the
monocyte differentiation marker and LPS receptor CD14, because HBP is
known to bind to the lipid A moiety of LPS (14, 15). However,
preincubation of whole blood with the anti-CD14 mAb (MY4, 20
µg/ml, 60 min at 37°C) did not change FITC-HBP binding to monocytes
over a period of 180 min (Fig. 5,
A and B).
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Effect of LPS.
LPS (10 µg/ml) significantly reduced early FITC-HBP affinity to
monocytes (after 1 to 15 min) but not at later time points (30 and 60
min) (Fig. 6A, solid
squares).
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, A and B).
Effect of temperature.
The binding of HBP to monocytes could be mediated by mechanisms
requiring energy for receptor presentation or HBP internalization.
Indeed, our results (Fig. 6C) demonstrated that
FITC-HBP had no affinity to monocytes at 4°C; only 3 ± 1% of
monocytes (n = 5 donors) were fluorescent (Fig. 7), with a fluorescence intensity of
21 ± 1, a value that corresponds to cellular autofluorescence.
However, at 37°C, 89 ± 6% monocytes (n
= 5 donors) were fluorescent (Fig. 7), with a fluorescence intensity of
144 ± 16 (p < .0001, mean ± SEM).
In contrast, changes in temperature from 37°C to 4°C did not change
the binding of Mo2 (anti-CD14 mAb) or FITC-LPS to monocytes (Fig. 7). These results demonstrated completely different binding properties
of LPS and HBP to human monocytes.
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In separate experiments, monocytes were isolated using
1-Step-Monocyte, a method that yields a 90% pure monocyte population.
Confocal microscopy was used to localize fluorescence from FITC-HBP in
monocytes. Experiments performed over a period of 15 to 240 min
demonstrated fluorescence on the cell surface at 15 min and within the
monocytes by 30 min (Fig. 8A). To show that HBP
was localized within CD14-positive monocytes, we incubated monocytes
for 60 min with FITC-HBP and labeled CD14 on the monocyte membrane with
Mo2-PE (25 min at 4°C). The use of two filters during confocal
microscopy (530 nm for FITC and 580 nm for PE) allowed us to assess
both FITC and PE fluorescence within the same monocyte. The results
confirmed the findings of the initial studies and demonstrated
FITC-fluorescence inside a ring of red-labeled monocyte membranes (Fig. 8B).
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Because HBP binding was not altered by blockade of CD14 or CD18,
HBP could have an effect on CD14 or CD18 expression as a mechanism to
increase monocyte production of inflammatory mediators. In these
experiments, HBP was incubated with monocytes at three different
concentrations (0.1, 1, and 10 µg/ml). HBP did not change CD14 (Fig. 9A) or CD18 (Fig. 9B) expression at any of the concentrations used.
However, the expression of the CD14 was altered by LPS (Fig. 10). After 4 h of incubation with 10 µg/ml of
LPS, there was a small but significant decrease in the number of cells
expressing CD14 (Fig. 10A). The number of cells
expressing CD14 then continued to decrease over the 24-h experiment.
There was also an increase in the MFI at 1 and 4 h after
incubation with 10 µg/ml LPS (Fig. 10B), indicating
an increased expression of CD14 per cell. Longer incubation times (18
and 24 h), however, resulted in a decreased MFI or a reduction in
the number of CD14 expressed per cell. In a separate experiment, we
demonstrated that HBP did not further modulate the LPS-induced changes
in monocyte CD14 expression after 4 and 24 h
(n = 5 donors; data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and that HBP
increases the LPS-induced release of TNF- and PGE2
(Fig. 2). Our results demonstrate that HBP, when compared with
lymphocytes or granulocytes, has a high affinity to CD14-positive
monocytes (Figs. 3 and 4) but not to the LPS receptor CD14 (Fig. 5).
Furthermore, we demonstrate that HBP is internalized in monocytes (Fig. 8), and that HBP increases the production of proinflammatory mediators
by mechanisms other than increased monocyte CD14 or CD18 expression
(Fig. 9).
We found that HBP induced TNF- release in the absence of LPS. These
findings differ from the results reported by Rasmussen et al. (12).
Those authors report that HBP has a potentiating effect on LPS-induced
cytokine production. They found that 20 µg/ml of HBP and,
surprisingly, 10 ng of LPS alone did not increase the release of IL-1,
IL-6, or TNF- from isolated monocytes (12), whereas we found large
TNF- responses at lower doses of HBP and the same dose of LPS.
Interestingly, three donors in our experiment (Fig. 1) were very low
responders to HBP and low responders to LPS. Rasmussen et al. (12)
might have been looking at a single low responding individual. The
variability in the current experiments in HBP responsiveness (Fig. 1)
could be similar to the variability for LPS-induced TNF- production,
which depends on genetic factors (22, 23). While the data suggest that
the sensitivity to HBP-induced TNF- production is also genetically
determined, we found no evidence of a sex-linked sensitivity. There was
also the possibility that the variability in our data and/or the
positive response to HBP alone could be due to LPS contamination. We
measured the LPS concentration in the HBP stock solution and calculated
a final contamination of 12 to 24 pg/ml LPS in the culture medium.
However, our LPS dose-response study (Fig. 1A)
demonstrated that a 100-fold higher concentration of LPS (1.25 ng/ml)
did not by itself produce a significant increase in TNF- production.
This indicates that HBP, and not LPS contamination, dramatically
increased the TNF- production that was induced by 10 ng/ml of LPS
(Fig. 2).
LPS activates monocytes via membrane-bound CD14 on monocytes (16, 24), an effect that is increased in the presence of the serum protein LBP (18). The functional LPS receptor appears to be a multimeric receptor that consists of the glycosylphosphatidyl-anchored CD14 and a presently unidentified transmembrane protein (16). Interestingly, substances other than LPS, such as uronic acid polymers (25) or other bacterial cell wall products (such as soluble peptidoglycans (26) or lipoarabinomannan from Mycobacterium tuberculosis (27)), induce cytokine production in monocytes via CD14.
These multimeric properties of the LPS receptor suggest that HBP could
act through CD14 to activate monocytes and release TNF- and
PGE2 (Fig. 2). This idea was strengthened by the fact that
HBP had a high affinity to monocytes (Figs. 3 and 4). Therefore, we
investigated whether HBP binds to CD14 on monocytes. Inhibition of the
interaction of LPS-LBP with CD14 can be achieved with a number of mAbs,
including the broadly reactive MY4 (28). We used 20 µg/ml of MY4 to
block CD14, a concentration that completely inhibited FITC-LPS binding
to CD14 (29). However, MY4 did not induce a reduction of HBP binding to
monocytes (Fig. 5). Therefore, we conclude that HBP does not bind to
the CD14 epitope recognized by MY4. This is further supported by the
fact that both LPS and anti-CD14 mAbs bind to constitutively
expressed CD14 on monocytes at 4°C, whereas HBP does not bind to
monocytes at 4°C (Fig. 7).
Others have shown that the number of membrane-bound CD14 correlates
with the production of LPS-induced cytokines such as IL-8 (30). We
investigated the modulation of monocyte CD14 expression by HBP and LPS
(Figs. 9 and 10). Our results showed that HBP did not modulate monocyte
CD14 expression, suggesting that up-regulation of CD14 is not the
mechanism responsible for the effect of HBP on the enhancement of
LPS-induced TNF- release. However, LPS induced a biphasic pattern of
monocyte CD14 expression (Fig. 10), with up-regulation of CD14 at
4 h and down-regulation of CD14 at 18 and 24 h. The early
up-regulation of monocyte CD14 was also reported by Marchant et al.
(31), who postulated a translocation of CD14 from an intracellular pool
to the cell surface. A down-regulation of CD14 at 18 h was
described by Wright (32) and by Landmann et al. (33). However, a 2-day
incubation of LPS caused increased levels of CD14 mRNA, membrane-bound
CD14, and soluble CD14 (33). The authors concluded that CD14 is
transcriptionally up-regulated by LPS (33). Studies by Bazil and
Strominger (34) showed that LPS down-regulates monocyte CD14 expression
by shedding the receptor from the surface. In our experiments, HBP
binding to monocytes was not inhibited with CD14 blockade (Fig. 5), and
HBP did not alter monocyte CD14 expression when compared with the
control group (Fig. 9). In addition, in the current experiments, the
binding characteristics of LPS and HBP are radically different, since
binding of FITC-HBP to monocytes was completely inhibited at 4°C, but
binding of FITC-LPS was not (Fig. 7). Therefore, it does not appear
that the effect of HBP can be attributed to an effect on the LPS
receptors CD14 or CD18.
Initially, binding of HBP to monocytes was reduced in the presence of
LPS, but there was no significant difference after 30 min (Fig. 6A). We therefore tested the impact of different
sequential additions of LPS and HBP and found no differences in TNF-
production after 24 h. This indicates that the initial binding of
HBP and LPS to monocytes does not alter the functional effect of HBP on
the enhancement of LPS-induced TNF- production. We propose that HBP,
inside the cell (Fig. 8), activates intracellular pathways, with the
potential to increase the LPS-induced signaling cascade derived from
CD14. This hypothesis of intracellular influence on signaling by HBP is
supported by the recent study of Pereira (35), who demonstrated that
HBP regulates vascular endothelial cell protein kinase C in both a
time- and dose-dependent manner.
Campbell (36) used two radiolabeled serine proteases with homology to
HBP, leukocyte elastase, and cathepsin G, and also radiolabeled
lactoferrin (another glycoprotein released from neutrophil granules),
to study binding to alveolar macrophages. Saturable binding of all
three proteins at 0°C was described, and the three proteins bound to
a similar number (54–73 x 106) of sites per cell.
Campbell argued that these receptors would be ideally suited to clear
neutrophil granule contents from the extracellular space in inflamed
tissues (36). Our affinity studies of HBP to human monocytes
demonstrate a lack of binding at 4°C (Fig. 7) and argue for an
energy-requiring step such as receptor turnover or HBP internalization.
In contrast to Campbell’s "clearance" hypothesis, we showed that
the binding and internalization of HBP to monocytes was concurrent with
an increased TNF- and PGE2 production and appeared to
be, at least, additive to the effects of LPS on the production of these
two mediators. These findings suggest that the binding and
internalization of free HBP is not only a clearance phenomenon, but may
represent an inducer and possibly an amplifier phenomenon of monocyte
activation.
CD18 is also a possible LPS receptor (17) that binds particulate LPS
but is unnecessary for the responses of macrophages to LPS (24).
Blocking of CD18 with the anti-CD18 Ab IB4 did not inhibit HBP
binding to monocytes in our experiments (Fig. 9). Wright et al. (24)
demonstrated that blocking CD18 did not reduce LPS-induced TNF-
synthesis. These findings indicate that CD18 is not an important
receptor in the HBP-mediated increase of monocyte-derived inflammatory
mediators and is not a receptor for HBP.
To date, the mechanism by which HBP induces the release of TNF- and
PGE2 and increases the LPS-induced release of these two
proinflammatory mediators in monocytes remains unknown. However, we
have demonstrated that the mechanism does not include binding to CD14
or modulation of CD14 expression on monocytes, which is contrary to our
original hypothesis. The lack of HBP binding to monocytes at 4°C
argues for an energy-dependent step in this process that may consist of
internalization of HBP.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Michael Heinzelmann, M.D., Ph.D., c/o M. Abby, Editorial Office, Department of Surgery, University of Louisville, Louisville, KY 40292. E-mail address:
3 Abbreviations used in this paper: HBP, heparin-binding protein; LBP, LPS-binding protein; FSC, forward scatter; SSC, sideways scatter; MFI, mean fluorescence intensity; PE, phycoerythrin; Mo2, anti-CD14 mAb.
Received for publication July 25, 1997. Accepted for publication February 5, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 T. D. Lee, M. L. Gonzalez, P. Kumar, S. Chary-Reddy, P. Grammas, and H. A. Pereira CAP37, a Novel Inflammatory Mediator : Its Expression in Endothelial Cells and Localization to Atherosclerotic Lesions Am. J. Pathol., March 1, 2002; 160(3): 841 - 848. [Abstract] [Full Text] [PDF]
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H. Tapper, A. Karlsson, M. Morgelin, H. Flodgaard, and H. Herwald Secretion of heparin-binding protein from human neutrophils is determined by its localization in azurophilic granules and secretory vesicles Blood, March 1, 2002; 99(5): 1785 - 1793. [Abstract] [Full Text] [PDF]
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O. Levy Antimicrobial proteins and peptides of blood: templates for novel antimicrobial agents Blood, October 15, 2000; 96(8): 2664 - 2672. [Abstract] [Full Text] [PDF]
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M. Heinzelmann, A. Platz, H. Flodgaard, H. C. Polk Jr., and F. N. Miller Endocytosis of Heparin-Binding Protein (CAP37) Is Essential for the Enhancement of Lipopolysaccharide-Induced TNF-{alpha} Production in Human Monocytes J. Immunol., April 1, 1999; 162(7): 4240 - 4245. [Abstract] [Full Text] [PDF]
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M. Heinzelmann, H. C. Polk Jr., and F. N. Miller Modulation of Lipopolysaccharide-Induced Monocyte Activation by Heparin-Binding Protein and Fucoidan Infect. Immun., December 1, 1998; 66(12): 5842 - 5847. [Abstract] [Full Text] [PDF]
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 B. J. Prendergast, D. A. Freeman, I. Zucker, and R. J. Nelson Periodic arousal from hibernation is necessary for initiation of immune responses in ground squirrels Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2002; 282(4): R1054 - R1062. [Abstract] [Full Text] [PDF]
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