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Department of Pathology University of Michigan Medical School, Ann Arbor, MI 48109; and
Department of Immunology, Genentech, South San Francisco, CA 94080
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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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In our original study of CLP-induced sepsis, the protective effects of anti-C5a on survival were documented (14), but evidence of MOF and the effects of anti-C5a on MOF were not assessed. In the current studies, we describe the progressive onset of MOF in the CLP model and the multifunctional loss of chemotactic responsiveness of blood neutrophils. We demonstrate the ability of anti-C5a to preserve chemotactic responsiveness of neutrophils and to preserve C5a binding sites on neutrophils, all of which are associated with greatly reduced development of MOF and lactate acidosis following CLP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unless otherwise specified, chemicals and reagents were purchased from Sigma (St. Louis, MO).
Preparation and characterization of Ab against rat C5a
Rat C5a peptide with the sequence KHRVPKKCCYDGARENKYET (corresponding to amino acid residues 17–36) was coupled to keyhole limpet hemocyanin and then used as an Ag to immunize rabbits. After several immunizations, the Ab was affinity purified from serum using the synthetic peptide coupled to beads. The preparation of this Ab was performed by Research Genetics (Huntsville, AL). In vitro, this polyclonal Ab immunoprecipitated from activated rat serum a protein aligning in Western blots with the 14-kDa marker, consistent with the molecular mass of glycosylated C5a (data not shown). This Ab did not interfere with whole hemolytic activity (CH50) in rat serum (data not shown).
Preparation of rat recombinant C5a (rr C5a)
A ‘his tag’ based on the TAGZyme system (Unizyme Laboratories, New York, NY) was engineered into the amino terminus of the rat C5a sequence. The presence of the alternating histidine and glutamine residues (in all six, each alternating in tandem) facilitated purification of the recombinant C5a protein over a Ni2+ Sepharose column. The his-tagged sequence was cloned into the baculovirus vector pVL1393 (PharMingen, San Diego, CA) and expressed in Spodoptera frugiperda (Sf9) cells (PharMingen). Recombinant protein was then purified by lysing the cells in buffer containing 10 mM Tris pH 7.4, 130 mM NaC1, 1% Triton X-100, 10 mM NaF, 10 mM NaH2PO4, 10 mM sodium pyrophosphate, and protease inhibitors (16 µg/ml benzamidine HCl, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). After removing the cell debris by centrifugation (10,000 x g for 10 min, then 100,000 x g for 1 h), the lysate was poured over a Ni2+ column (Qiagen, Chatsworth, CA). Then, rr C5a was eluted with 10 mM Tris (pH 7.4), 300 mM NaCl, and 0–250 mM imidazole. The eluate was dialyzed extensively with PBS (pH 7.4). This rr C5a was shown to be functionally active in a chemotaxis assay using rat blood neutrophils.
Experimental sepsis by CLP
Male Long-Evans specific pathogen-free rats (Harlan Breeders, Indianapolis, IN) weighing 275–300 g were used in all experiments. Anesthesia was induced by i.p. administration of ketamine (20 mg/100 g body weight). After shaving the abdomen and applying a topical disinfectant, a 2-cm midline incision was made, and the cecum was identified and ligated below the ileocecal valve, with care being taken not to occlude the bowel passage. The cecum was then subjected to a single "through and through" perforation with a 21-gauge needle and was gently squeezed to ensure patency of the perforation sites. After repositioning the bowel, the abdominal incision was closed in layers with plain gut surgical suture 4-0 and skin clips (Ethicon, Somerville, NJ). Sham-operated animals underwent the same procedure except for ligation and puncture of the cecum. Animals with CLP treatment were injected i.v. immediately after induction of CLP with 400 µg rabbit IgG against rat C5a (anti-C5a) or with 400 µg preimmune rabbit IgG. Before and after surgery, animals had unrestricted access to food and water.
For urine collection and determination of water intake and urinary output, rats were closely followed in metabolic boxes (Nalgene metabolic cage; Nalge Nunc International, Rochester, NY) from time 0–36 h after the surgical procedures, as indicated in the individual experiments.
In some animals, a carotid artery catheter (PE-50; Becton Dickinson, Sparks, MD) was placed through an anterior cervical incision followed by s.c. tunneling and externalization in the posterior cervical area. Unless otherwise indicated, arterial blood samples were collected at 12-h intervals after CLP to measure blood lactate, pH, pO2, pCO2, and HCO3- using a blood gas analyzer (OSM3, ABL 505, and EML 100; Radiometer, Copenhagen NV, Denmark). In other groups of rats, blood was obtained from the inferior vena cava (at 12-h intervals up to 48 h after CLP) for biochemical measurements and for isolation of neutrophils. Organs were processed for morphological studies as described below.
Transmission electron microscopy
Kidney tissues were sectioned into 1-mm3 cubes, fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) at room temperature overnight, and washed in two changes of 0.1 M cacodylate buffer (pH 7.3). Samples were postfixed in 2% OsO4 in 0.1 M cacodylate buffer (pH 7.3) for 1 h at 25°C, dehydrated in graded series of alcohols for final dehydration in propylene oxide, infiltrated with increasing mixtures of propylene oxide and Epon resin, and embedded in pure Epon. One-micron sections stained with toluidine blue were evaluated by light microscopy. Thin sections were obtained on an AO Ultracut ultramicrotome, stained with uranyl acetate and lead citrate, and evaluated in a Phillips 400 T electron microscope.
Measurement of biochemical parameters
Unless otherwise stated, blood was obtained from the inferior vena cava at the time of sacrifice. Creatinine, blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were determined in serum samples by standard clinical chemistry techniques. All assays were performed using commercially available reagents (Vitros dry film slides) on a Vitros 950 chemistry analyzer (Ortho-Clinical Diagnostics, Rochester, NY). Electrolytes were determined by conventional flame spectrometry.
Calculation of glomerular filtration rates (GFRs)
The GFR was determined by endogenous creatinine clearance, measured by the urine flow rate (ml/min) over a period of 36 h, and multiplied by the ratio of the creatinine concentration in urine and in serum.
Urinary protein electrophoresis
After centrifugation of urine at 14,000 x g for 10 min, a 10-µl aliquot of the supernatant was added to 5 µl of Laemmli’s sample buffer followed by boiling for 5 min. Samples were then electrophoresed on a 10% SDS-PAGE gel and stained with Coomassie blue.
Isolation of rat blood neutrophils
Whole blood from rats was drawn into syringes containing the anticoagulant ACD (Baxter Health Care, Mundelein, IL). Neutrophils were isolated using Ficoll-Paque gradient centrifugation (Pharmacia Biotech, Uppsala, Sweden) and dextran sedimentation. After hypotonic lysis of residual RBC, neutrophils were evaluated in chemotaxis assays and in binding studies using rr C5a and fMLP.
Chemotaxis assay
Following neutrophil isolation, cells were fluorescein labeled with BCECF (2',7'-bis [2-carboxyethyl]-5-[and 6]-carboxy-fluorescein acetoxymethyl ester) (Molecular Probes, Eugene, OR). Labeled neutrophils (5 x 106 cells/ml) were then loaded into the upper chambers of 96-well minichambers (Neuroprobe, Cabin John, MD). Lower chambers were loaded with the chemotactic peptides C5a or fMLP. The upper and lower chambers were separated by a polycarbonate membrane of 3-µm porosity (Neuroprobe). Minichambers were incubated for 30 min at 37°C. The number of cells migrating through polycarbonate filters to the lower surface was measured by cytofluorometry (Cytofluor II; PerSeptive Biosystems, Framingham, MA). Samples were measured in quadruplicate.
125I-rr C5a and [3H]fMLP binding studies
rr C5a (HQ]6C5a) was labeled with
125I, using the chloramine T-based method as
described (16). This protocol allows gentle oxidation,
which preserves the neutrophil chemotactic activity of C5a. Rat blood
neutrophils were obtained at 12, 24, 36, and 48 h after CLP and
analyzed for the ability of 125I-rr C5a to bind
to these cells. For reference purposes, blood neutrophils were also
obtained from otherwise nonmanipulated healthy rats. Briefly,
neutrophils (1 x 107 cells/ml) were
incubated at 4°C in assay buffer (HBSS, 0.1% BSA) in 1.5-ml
microcentrifuge tubes with 125I-rr C5a (specific
activity, 35 µCi/µg) in a final volume of 200 µl. After
incubation for 20 min at 4°C, cell suspensions were layered over 20%
sucrose and sedimented by centrifugation at 11,000 x g
in a swing-out rotor for 2 min. After centrifugation, the tubes were
frozen at -80°C and the tips (containing the cell pellet) were cut
off to determine the cell-bound 125I-rr C5a using
a gamma counter (1261 Multi
; Wallac, Gaithersburg, MD). Binding
affinities were calculated in the conventional manner
(17). Binding studies with
[3H]fMLP (0.1–100 nM) (specific activity, 96
Ci/mmol) (NEN, Boston, MA) were performed similarly. Cell-associated
[3H]fMLP was determined in a Beckman LS 5000 TD
liquid scintillation counter (Beckman, Palo Alto, CA). Specific binding
was calculated by subtracting nonspecific binding (in the presence of
100-fold excess of unlabeled fMLP; Sigma) from total binding.
Statistical analyses
All values are expressed as mean ± SEM. Data sets were analyzed with a one-way ANOVA. Individual group means were then compared using the Tukey test. Significance was assigned where p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Arterial blood gases (expressed as mmHg) and pH values were
determined in normal rats (ctrl) and in CLP rats that were infused i.v.
at time 0 (immediately after CLP) with either 400 µg rabbit preimmune
IgG or 400 µg anti-rat C5a IgG (anti-C5a). Blood samples were
obtained at early (24 h) and late time points (60 h) of CLP. The
results are shown in Table I
. As
expected, at 24 h there was a mild respiratory alkalosis, with the
arterial pH rising from 7.47 ± 0.01 to 7.53 ± 0.01 in CLP
rats treated with preimmune IgG. In the same group, the blood
pCO2 fell from a value of 38.6 ± 0.89 to
34.2 ± 0.91. The HCO3-
(mmol/L) and pO2 values were unchanged in these
two sets of animals at this early period of time. In contrast, in CLP
rats treated with anti-C5a, the blood pH and
pCO2 values at 24 h were the same as those
values obtained in normal, unmanipulated rats.
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). At the
same time point, first signs of an additional respiratory acidosis with
a mild hypercapnia were found (increase in blood
pCO2 from 36.6 ± 1.29 to 44.5 ± 3.32)
and there was clear evidence of hypoxia, with a fall in the
pO2 value from 105 ± 0.88 to 69.8 ±
9.79. Remarkably, in the CLP group treated with anti-C5a, all
values remained in the normal range at 60 h (Table I). Parameters of MOF and effects of anti-C5a treatment
The parameters of MOF are described in Table II as well as the protective effects of
anti-C5a. There was clear evidence of MOF developing in CLP rats
and the reversal of these parameters by treatment of CLP rats with
anti-C5a (400 µg given i.v. at the time of CLP) but not when
preimmune IgG was used. Blood lactate levels rose 2.64-fold during CLP.
This rise was prevented in anti-C5a-treated rats. Evidence of liver
dysfunction was indicated by elevations in serum bilirubin, ALT, and
AST (2.5-, 5-, and 7-fold, respectively). In animals treated with
anti-C5a, most of these parameters remained in the normal range,
whereas the rise in LDH levels was attenuated. With regard to kidney
function, in unprotected CLP animals, serum creatinine, BUN, and urine
protein levels rose by 67, 280, and 150%, respectively, whereas in CLP
rats treated with anti-C5a, the levels were not statistically
different from those levels in sham animals. Electrophoretic analysis
of urine (SDS-PAGE, followed by Coomassie blue staining) revealed faint
amounts of albumin in urine from shams and greatly increased urinary
albumin in CLP rats treated with preimmune IgG involving lower and
higher m.w. proteins relative to urinary albumin. In CLP rats treated
with anti-C5a, albumin was somewhat increased above that found in
sham rats, whereas the higher and lower m.w. bands were largely
abolished (data not shown). These data suggest defects both in
glomerular filtration as well as in tubular absorption. Urine output
and GFR values in the unprotected CLP animals fell by 80 and 75%,
respectively; these changes were greatly reduced in the animals
receiving anti-C5a. Thus, manifestations of MOF in CLP animals are
greatly attenuated in animals receiving anti-C5a.
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Given the changes in proteinuria, urine output, and GFR (see
above) we investigated presence of morphological changes in kidneys by
transmission electron microscopy. Tissue samples were obtained 36
h after CLP-induced sepsis in rats treated with either preimmune IgG or
anti-C5a. As shown in Fig. 1, in CLP
animals treated with preimmune IgG, it was evident that there was
extensive fusion of foot processes of podocytes in glomeruli
(A, arrowheads). In proximal convoluted tubules, the cells
had lost their cell membranes and exhibited mitochondrial swelling
together with intracellular edema (B). In CLP animals that
had been treated with anti-C5a, renal glomeruli appear normal with
prominent podocytes (C), and epithelial cells of proximal
convoluted tubules were intact and had normal structures
(D).
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The relative purity of recombinantly produced rr C5a was determined by electrophoretic analysis. Coomassie staining revealed a single predominant band in the expected m.w. position for a nonglycosylated product (data not shown) This C5a preparation was used both for functional (chemotaxis) assessment of rat blood neutrophils and binding of 125I-rr C5a to rat blood neutrophils.
Chemotactic dysfunction of blood neutrophils from CLP animals
The ability of blood neutrophils to respond in vitro to
recombinant rat C5a and to the structurally unrelated chemotactic
peptide, fMLP, both of which are known to be powerful chemotactic
factors for neutrophils, was assessed. Blood neutrophils were obtained
from each of the three groups of rats (control and 36 h after CLP
from rats treated with either preimmune IgG or anti-C5a), as
described above. The isolated cells were labeled with a fluorescent
probe and evaluated by cytofluorometry for chemotactic responses in
vitro as described in Materials and Methods. The results are
shown in Fig. 2. When blood neutrophils
were obtained from normal rats (ctrl), chemotactic responses to rr C5a
showed the expected bell-shaped dose-response profile, starting at 0.1
nM and peaking between 1 and 100 nM (Fig. 2A). Blood
neutrophils from CLP rats treated with preimmune IgG showed greatly
diminished chemotactic responsiveness over the entire range of
concentrations of rr C5a, whereas neutrophils obtained from CLP rats
treated with anti-C5a showed responses that were not statistically
different from those of blood neutrophils obtained from normal
rats.
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B.
Blood neutrophils from otherwise untreated rats showed the expected
pattern of dose response, progressing from 10-9
M fMLP and reaching a plateau between 10-6 and
10-4 M, followed by a decline at high
concentrations (10-3 M) of fMLP. Blood
neutrophils obtained from rats 36 h after CLP and treated with
preimmune IgG showed profoundly depressed chemotactic responsiveness
across the entire range of concentrations of fMLP (Fig. 2B,
lowest line). In striking contrast, blood neutrophils obtained from CLP
rats that had been treated with anti-C5a had chemotactic responses
that were indistinguishable from those obtained from blood of normal
animals. Thus, blood neutrophils from CLP rats acquired a chemotactic
defect that was nonspecific, involving impaired responses to both C5a
and fMLP. In vivo blockade of C5a in CLP rats resulted in retention of
normal chemotactic function in response to both C5a and fMLP. Binding of 125I- rr C5a to normal rat blood neutrophils
For binding studies, neutrophils were obtained as described above.
As shown in Fig. 3A, over a
range of 0.6–39.2 nM 125I-rr C5a, there was
progressively increased binding to neutrophils with a plateau in the
binding occurring between 19.6 and 31.4 nM
125I-rr C5a. When unlabeled rr C5a was mixed
together with 125I-rr C5a in a ratio of 100:1,
the binding of the latter was almost totally suppressed (Fig. 3A, lower line of graph). The computed
Kd value was 20 nM. For subsequent
studies, the dose of 31.4 nM 125I-rr C5a was used
to determine whether there was defective binding when blood neutrophils
from CLP rats were used.
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These studies were undertaken to assess the status of binding of
125I-rr C5a to blood neutrophils as a function of
time after onset of CLP. Neutrophils were isolated from blood at 0, 12,
24, 36, and 48 h after onset of CLP in animals that had been
treated i.v. at time 0 with 400 µg preimmune IgG or anti-C5a. The
results of these binding studies are shown in Fig. 3B.
Binding values of 
30,000 cpm with a nonspecific binding fraction of
5000 cpm (Fig. 3B) were found in blood neutrophils
obtained from normal rats (time 0). When CLP rats were pretreated with
400 µg preimmune IgG and blood neutrophils were harvested at 12, 24,
36, and 48 h after CLP, the specific binding values for rr C5a
relative to the 0 time values had fallen to 3.96, 4.35, 33.2, and
40.25%, respectively. In CLP animals that had been infused with
anti-C5a, the results for 12, 24, 36, and 48 h were
significantly different from that of preimmune IgG-treated rats.
Specific C5a binding values at these time points were 40.7, 61.2, 76.7,
and 92.3%, respectively. Thus, it appears that in CLP rats, treatment
with anti-C5a caused retention of the binding sites in blood
neutrophils. Whether this was due to occupancy by C5a of C5aR or due to
internalized C5a-C5aR complexes remains to be determined.
Binding of [3H]fMLP to rat blood neutrophils
Binding of [3H]fMLP to rat neutrophils
isolated from normal rat blood was determined as described above. As
seen in Fig. 4, there was a progressive
increase in binding over a range of 0.1–100 nM fMLP, reaching a
plateau at a concentration of 50 nM or higher
(Kd = 35 nM). Nonspecific
binding was determined by incubating neutrophils with a 100:1 ratio of
nonlabeled fMLP to [3H]fMLP. Neutrophils
obtained from normal rats or from CLP rats (24 h after CLP) treated
with either 400 µg preimmune IgG or 400 µg anti-C5a did not
show statistically significant differences in binding of
[3H]fMLP (50 nM). Thus, CLP does not reduce
fMLP binding in blood neutrophils, in striking contrast to the
situation with C5a binding (Fig. 3), even though the chemotactic
responses of fMLP are greatly suppressed (Fig. 2B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, CLP in rats caused a time-dependent pattern of MOF, with a late onset of hypoxia (60 h) in CLP rats treated with preimmune IgG but not in CLP rats after blockade of C5a. In a short-term sepsis model (4–6 h), pigs or monkeys challenged with very high doses of live Escherichia coli and treated with an Ab to porcine or human C5a showed (in comparison to otherwise untreated animals with sepsis) a partial improvement in some functional parameters (such as oxygenation, lung edema, and oxygen uptake) and an improvement in the oxygen extraction ratio accompanied by an attenuated increase in lactate levels (21, 22, 23). Hyperlactatemia is known to be a reliable indicator of advancing MOF in humans (24). In CLP animals treated with preimmune IgG, blood lactate levels were very high at 48 h. This elevation was abrogated in CLP rats treated with anti-C5a. Data in this report clearly indicate that CLP animals develop evidence of progressive lung, kidney, and liver failure that is C5a dependent. All of these outcomes may be linked to excessive C5a generation during sepsis, resulting in a compromised respiratory burst of neutrophils and their oxygen-dependent bactericidal pathways.
Our data also document in the CLP model in rats that blood neutrophils acquire a significant defect in chemotactic responsiveness both to C5a as well as to fMLP. Because these two chemotactic peptides are structurally totally distinct and use different receptors on neutrophils (25, 26), our observations may imply a postchemotactic factor receptor-acquired functional defect. This may be the result of excessive in vivo stimulation by C5a, resulting in defects in signal transduction that extend to agonists beyond C5a and impair chemotactic responsiveness to C5a as well as to other chemotactic factors. Our studies document development of chemotactic defects in blood neutrophils from CLP rats involving responses to both C5a and to fMLP. These defects are associated with loss of binding of rat 125I-rr C5a to neutrophils but normal binding of [3H]fMLP. fMLP is known to be the major neutrophil chemotactic factor in culture filtrates of E. coli (27). These data would support the possibility that in vivo contact of neutrophils with C5a induces a downstream defect in signal transduction beyond a point at which C5aR and fMLP-receptor (fMLPR) pathways merge. In other words, although blood neutrophils from CLP rats appear to demonstrate normal binding of fMLP, this binding is unable to effectively engage a post receptor signaling pathway that is common to both C5aR and fMLPR. This is consistent with earlier studies in which, when neutrophils were exposed to low levels of C5a (present in activated serum), the cells acquired a chemotactic defect specific to C5a. When neutrophils were exposed to higher concentrations of C5a, they then acquired a broad defect in chemotactic function characterized by loss of responsiveness to C5a and to bacterial chemotactic peptides (11). In vivo, the chemotactic responses of neutrophils obtained from patients with sepsis or multisystem trauma with adult respiratory distress syndrome were found to be specifically deactivated to C5a or deactivated to both C5a and fMLP depending on clinical severity and time after trauma or onset of sepsis. In contrast, no alteration in neutrophil function was found in patients who did not develop adult respiratory distress syndrome (28). Our recent studies in the CLP model indicate that the H2O2 response of neutrophils to PMA is impaired (14), suggesting a broad defect in postreceptor signaling pathways.
Perhaps relevant to the chemotactic dysfunction of blood neutrophils
described in this report is the finding that binding sites for rat C5a
(but not for fMLP) on neutrophils have been virtually lost (<5% of
binding in control neutrophils) during the first 24 h of sepsis.
By 48 h, neutrophils still demonstrated <50% of C5a binding, in
striking contrast to nearly normal binding values of C5a for
neutrophils obtained from CLP rats treated with anti-C5a (Fig. 3).
Under these latter conditions, chemotactic responsiveness to C5a and to
fMLP was totally preserved. These data are in accord with findings of
specific loss of C5a binding (but intact fMLP binding) to neutrophils
in patients with sepsis (11). It seems possible that the
mechanism by which anti-C5a may protect against development of MOF
during sepsis is its ability to cause preservation in the chemotactic
and respiratory burst function of blood neutrophils by intercepting C5a
before it has a chance to bind to C5aR on neutrophils. Retention of
these functional responses would facilitate effective in vivo
recruitment of neutrophils and maintenance of bactericidal function in
these cells. Understanding the relationship between C5a and the
induction of host defense dysfunction in blood neutrophils is the topic
of current focus.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter A. Ward, Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, Ann Arbor, MI 48109-0602.
3 Abbreviations used in this paper: MOF, multiorgan failure; CLP, cecal ligation/puncture; rr, rat recombinant; GFR, glomerular filtration rate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; LDH, lactate dehydrogenase.
Received for publication August 25, 2000. Accepted for publication October 16, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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I. Farkas, M. Takahashi, A. Fukuda, N. Yamamoto, H. Akatsu, L. Baranyi, H. Tateyama, T. Yamamoto, N. Okada, and H. Okada Complement C5a Receptor-Mediated Signaling May Be Involved in Neurodegeneration in Alzheimer's Disease J. Immunol., June 1, 2003; 170(11): 5764 - 5771. [Abstract] [Full Text] [PDF]
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N. C. Riedemann, T. A. Neff, R.-F. Guo, K. D. Bernacki, I. J. Laudes, J. V. Sarma, J. D. Lambris, and P. A. Ward Protective Effects of IL-6 Blockade in Sepsis Are Linked to Reduced C5a Receptor Expression J. Immunol., January 1, 2003; 170(1): 503 - 507. [Abstract] [Full Text] [PDF]
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M. S. HUBER-LANG, N. C. RIEDEMAN, J. V. SARMA, E. M. YOUNKIN, S. R. McGUIRE, I. J. LAUDES, K. T. LU, R.-F. GUO, T. A. NEFF, V. A. PADGAONKAR, et al. Protection of innate immunity by C5aR antagonist in septic mice FASEB J, October 1, 2002; 16(12): 1567 - 1574. [Abstract] [Full Text] [PDF]
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M. S. Huber-Lang, E. M. Younkin, J. V. Sarma, S. R. McGuire, K. T. Lu, R. F. Guo, V. A. Padgaonkar, J. T. Curnutte, R. Erickson, and P. A. Ward Complement-Induced Impairment of Innate Immunity During Sepsis J. Immunol., September 15, 2002; 169(6): 3223 - 3231. [Abstract] [Full Text] [PDF]
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 T. E. Mollnes, O.-L. Brekke, M. Fung, H. Fure, D. Christiansen, G. Bergseth, V. Videm, K. T. Lappegard, J. Kohl, and J. D. Lambris Essential role of the C5a receptor in E coli-induced oxidative burst and phagocytosis revealed by a novel lepirudin-based human whole blood model of inflammation Blood, August 13, 2002; 100(5): 1869 - 1877. [Abstract] [Full Text] [PDF]
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R.-F. Guo, N. C. Riedemann, I. J. Laudes, V. J. Sarma, R. G. Kunkel, K. A. Dilley, J. D. Paulauskis, and P. A. Ward Altered Neutrophil Trafficking During Sepsis J. Immunol., July 1, 2002; 169(1): 307 - 314. [Abstract] [Full Text] [PDF]
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 I. J. Laudes, J. C. Chu, S. Sikranth, M. Huber-Lang, R.-F. Guo, N. Riedemann, J. V. Sarma, A. H. Schmaier, and P. A. Ward Anti-C5a Ameliorates Coagulation/Fibrinolytic Protein Changes in a Rat Model of Sepsis Am. J. Pathol., May 1, 2002; 160(5): 1867 - 1875. [Abstract] [Full Text] [PDF]
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N. C. Riedemann, R.-F. Guo, V. J. Sarma, I. J. Laudes, M. Huber-Lang, R. L. Warner, E. A. Albrecht, C. L. Speyer, and P. A. Ward Expression and Function of the C5a Receptor in Rat Alveolar Epithelial Cells J. Immunol., February 15, 2002; 168(4): 1919 - 1925. [Abstract] [Full Text] [PDF]
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H.-K. Yum, J. Arcaroli, J. Kupfner, R. Shenkar, J. M. Penninger, T. Sasaki, K.-Y. Yang, J. S. Park, and E. Abraham Involvement of Phosphoinositide 3-Kinases in Neutrophil Activation and the Development of Acute Lung Injury J. Immunol., December 1, 2001; 167(11): 6601 - 6608. [Abstract] [Full Text] [PDF]
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 T. S. Olson, K. Singbartl, and K. Ley L-selectin is required for fMLP- but not C5a-induced margination of neutrophils in pulmonary circulation Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2002; 282(4): R1245 - R1252. [Abstract] [Full Text] [PDF]
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) or anti-rat C5a (anti-C5a) (
).
Nonspecific binding was determined by binding in the presence of
100-fold excess of cold fMLP (
).