?-1-Antitrypsin is an endogenous inhibitor of proinflammatory
cytokine production in whole blood
Gregory B. Pott,*,†Edward D. Chan,*,†,‡Charles A. Dinarello,†and Leland Shapiro*,†,1
*Denver Veterans Affairs Medical Center,†University of Colorado Denver, and‡National Jewish Medical and
Research Center, Denver, Colorado, USA
nous suppressors of inflammatory mediators are
present in human blood. ?-1-Antitrypsin (AAT) is
the most abundant serine protease inhibitor in
blood, and AAT possesses anti-inflammatory activ-
ity in vitro and in vivo. Here, we show that in vitro
stimulation of whole blood from persons with a
genetic AAT deficiency resulted in enhanced cyto-
kine production compared with blood from healthy
subjects. Using whole blood from healthy subjects,
dilution of blood with RPMI tissue-culture me-
dium, followed by incubation for 18 h, increased
spontaneous production of IL-8, TNF-?, IL-1?,
and IL-1R antagonist (IL-1Ra) significantly, com-
pared with undiluted blood. Dilution-induced cyto-
kine production suggested the presence of one or
more circulating inhibitors of cytokine synthesis
present in blood. Serially diluting blood with tissue-
culture medium in the presence of cytokine stimu-
lation with heat-killed Staphylococcus epidermidis
(S. epi) resulted in 1.2- to 55-fold increases in
cytokine production compared with S. epi stimula-
tion alone. Diluting blood with autologous plasma
did not increase the production of IL-8, TNF-?,
IL-1?, or IL-1Ra, suggesting that the endogenous,
inhibitory activity of blood resided in plasma. In
whole blood, diluted and stimulated with S. epi,
exogenous AAT inhibited IL-8, IL-6, TNF-?, and
IL-1? significantly but did not suppress induction
of the anti-inflammatory cytokines IL-1Ra and IL-
10. These ex vivo and in vitro observations suggest
that endogenous AAT in blood contributes to the
suppression of proinflammatory cytokine synthesis.
J. Leukoc. Biol. 85: 000–000; 2009.
Several observations suggest endoge-
Key Words: inflammation ? dilution ? serine protease inhibitor
Numerous observations demonstrate the presence of endoge-
nous substances in blood, which suppresses the synthesis of
proinflammatory molecules. For example, in health, biologi-
cally active, proinflammatory cytokines are rarely detected in
blood . However, diluting whole blood enhances proinflam-
matory cytokine production in vitro, suggesting that dilution
reduces the function of cytokine inhibitors in blood. Chernoff
et al.  reported that whole blood dilution enhanced IL-1?
and TNF-? production. Similarly, synthesis of intracellular
IFN (IFN-?), TNF-?, IL-2, and IL-10 was optimized when
blood was diluted in tissue-culture medium [3–6]. Therefore,
diluting whole blood likely reduces concentrations of circulat-
ing inhibitors of cytokine production and increases cytokine
synthesis per cell. Despite the common practice of diluting
blood to enhance cytokine production, the identity of circulat-
ing inhibitors remains unclear.
?-1-Antitrypsin (AAT) is a 394-aa, 52-kDa glycoprotein
synthesized primarily by hepatocytes , with smaller amounts
synthesized by intestinal epithelial cells, neutrophils, pulmo-
nary alveolar cells, and macrophages [8, 9]. AAT is the most
abundant, endogenous serine protease inhibitor (Pi) in the
circulation. Serum AAT concentrations in healthy subjects are
1.5–3.5 mg/mL and can increase fourfold during inflammation,
indicating that AAT is an acute-phase protein [7, 10, 11]. The
primary function of AAT is thought to be inactivation of neu-
trophil elastase and other endogenous serine proteases.
AAT has been studied extensively in the clinical setting as
a result of the existence of genetic defects, resulting in abnor-
mally low AAT concentrations in blood and referred to as AAT
deficiency. More than 100 AAT alleles have been described,
and the normal M-type AAT protein is designated PiM .
Persons with normal AAT concentrations in blood usually have
two copies of the PiM gene (PiMM phenotype), and the prev-
alence of this phenotype is ?83% in the United States popu-
lation . Reduced AAT levels are typically associated with
Z-type (PiZ) or S-type (PiS) AAT variants. The term “AAT
deficiency” often refers to the presence of homozygous PiZZ
AAT, where serum AAT levels approximate 10–15% of normal
. However, other AAT phenotypes are associated with
deficient AAT levels and include PiSS, PiSZ, and the pairing
of PiZ or PiS with the normal PiM protein (PiMZ or PiMS) .
AAT-deficient individuals are at increased risk for extensive
and early onset pulmonary emphysema thought to result from
progressive destruction of alveolar walls as a result of unop-
posed activity of neutrophil-derived elastase .
1Correspondence: Denver Veterans Affairs Medical Center and Department
of Medicine, Division of Infectious Diseases, University of Colorado Denver,
12700 E. 19th Ave., Box B168, Aurora, CO 80045, USA. E-mail:
Received February 28, 2008; revised December 16, 2008; accepted January
0741-5400/09/0085-0001 © Society for Leukocyte Biology
Journal of Leukocyte Biology
Volume 85, May 2009
Uncorrected Version. Published on February 5, 2009 as DOI:10.1189/jlb.0208145
Copyright 2009 by The Society for Leukocyte Biology.
Novel studies have expanded the link between AAT and
human disease. For example, associations were shown between
reduced AAT levels or abnormal AAT proteins and HIV type
1 infection [16–18], hepatitis C infection and chronic liver
disease , atypical mycobacterial infection , diabetes
mellitus , and panniculitis . In mice, exogenous AAT
protected islet cell allografts from rejection , blocked ? cell
apoptosis , prevented pulmonary emphysema , and
inhibited angiogenesis and tumor growth .
Adding AAT to cultured human cells in vitro has revealed
anti-inflammatory properties. For example, Janciauskiene et al.
 reported that AAT inhibited LPS-stimulated synthesis and
secretion of TNF-? and IL-1? in human blood monocytes. In
addition, intracellular signaling studies showed that AAT in-
hibited activation of NF-?B, a transcription factor involved in
the expression of several proinflammatory cytokines . Im-
portantly, some reports suggest that the anti-inflammatory
properties of AAT may not require the serine Pi activity of AAT
[15, 28, 29].
Investigations of AAT anti-inflammatory functions have used
cell lines or PBMC-derived macrophages. As these experimen-
tal designs may not reflect AAT biological activity in vivo, we
used an in vitro assay of cultured human whole blood to
conduct studies that represent in vivo conditions more closely.
In the present investigations, we addressed several issues in
cytokine biology. First, in whole blood cultures, we compared
cytokine synthesis in subjects with genetic AAT deficiency
with cytokine synthesis in healthy subjects. Second, in whole
blood from healthy subjects, we examined the effect of blood
dilution on constitutive (spontaneous) and on Staphylococcus
epidermidis (S. epi)-stimulated cytokine synthesis. Third, we
compared spontaneous cytokine production in whole blood
diluted with tissue-culture medium with blood diluted with
autologous plasma. Fourth, we evaluated exogenously added
AAT as an inhibitor of cytokine production in diluted and S.
epi-stimulated whole blood cultures. Finally, a synthetic serine
Pi was studied for effects on whole blood cytokine production.
MATERIALS AND METHODS
S. epi strain 49134 was obtained from the American Type Culture Collection
(Remel, Lenexa, KS, USA) and provided by Dr. Mary Bessesen (Denver
Veterans Affairs Medical Center and the University of Colorado Denver, CO,
USA). S. epi was grown overnight in suspension cultures in Luria-Bertani
medium (Difco, Detroit, MI, USA). The S. epi suspension was heat-killed by
boiling for 30 min and the protein concentration of the heat-killed S. epi
preparation determined using the Coomassie Plus protein assay reagent
(Pierce, Rockford, IL, USA). Clinical-grade human AAT (Aralast?, 20 mg/mL
stock solution, Baxter Healthcare Corp., Westlake Village, CA, USA) and
clinical-grade human serum-derived albumin (250 mg/mL stock solution, ZLB
Bioplasma AG, Berne, Switzerland) were used in these studies. H-Ala-Ala-
Pro-Val-chloromethylketone (AAPV-CMK) was obtained from Bachem (King
of Prussia, PA, USA) and solubilized in DMSO (Fisher Biotech, Fair Lawn, NJ,
USA) at a stock concentration of 50 mM. RPMI-1640 medium and PBS were
purchased from Mediatech (Herndon, VA, USA), and HBSS was obtained from
Life Technologies (Grand Island, NY, USA).
Whole blood collection from healthy volunteers
Healthy subjects not taking prescribed or over-the-counter medications par-
ticipated. Blood obtained following antecubital venipuncture was aspirated
into sterile glass vacuum tubes containing freeze-dried sodium heparin that
resulted in a final heparin concentration of 14.3 units/mL (Becton Dickinson,
Franklin Lakes, NJ, USA). Informed consent was obtained from each subject,
and the Human Subject Institutional Review Board at the University of
Colorado Denver approved the protocol.
Whole blood collection from AAT-deficient
Nine patients with AAT deficiency were studied. AAT deficiency was estab-
lished using criteria defined previously, including serum AAT levels below
0.72 mg/mL (prior to initiation of replacement therapy) and the presence of
Z-type AAT mutant protein by phenotype analysis. Five patients had the PiZZ
mutant phenotype, two had the PiSZ phenotype, and two had the PiMZ
phenotype. Eight of the nine AAT-deficient patients were treated with weekly
i.v. infusions of 60 mg/kg Prolastin?(Talecris Biotherapeutics, Research
Triangle Park, NC, USA), and one patient was treated with weekly infusions of
60 mg/kg Zemaira?(Aventis Behring LLC, King of Prussia, PA, USA). All nine
AAT-deficient patients were diagnosed with chronic obstructive pulmonary
disease and treated with inhaled fluticasone-salmeterol, and eight of the nine
patients were treated with inhaled albuterol. Other prescribed medications
included fluoxetine, tiotropium, iprotropium, lansoprazole, atorvastatin, alen-
dronate, and fexofenadine. AAT concentrations from AAT-deficient and
healthy volunteers were determined in heparanized plasma using a human
AAT quantitative ELISA (GenWay Biotech, Inc., San Diego, CA, USA).
Informed consent was obtained from each AAT-deficient subject, and the
Human Subject Institutional Review Board at the National Jewish Medical and
Research Center (Denver, CO, USA) approved the protocol.
Cytokine production in whole blood cultures
from AAT-deficient persons and healthy
Heparinized venous blood obtained from healthy volunteers (controls) and from
AAT-deficient donors was transferred to 12 ? 75 mm snap-cap polypropylene
tubes (Becton Dickinson) under sterile conditions. Heat-killed S. epi (final
protein concentration, 1.2 ?g/mL) was added to undiluted whole blood (1.0 mL
final vol), and the cultures incubated with caps loosely applied at 37°C and 5%
CO2for 18 h. After incubation, the culture supernatants were aspirated,
transferred to new tubes, and frozen at –70°C until assayed.
Whole blood dilution studies
Freshly obtained whole blood from each of four healthy subjects was aliquoted
into 12 ? 75 mm snap-cap polypropylene tubes. The blood from each subject
was cultured without dilution or was serially diluted with RPMI to 1:4, 1:8,
1:16, or 1:32 in the absence or presence of 1.2 ?g/mL (final concentration) S.
epi in a volume of 1.0 mL. The whole blood cultures were then incubated as
Whole blood diluted with RPMI or autologous
Donor-matched (autologous) plasma was obtained by subjecting 8.0 mL hep-
arinized blood from each of four healthy volunteers to 400 g centrifugation for
10 min and aspirating the supernatant plasma. Whole blood was diluted 1:32
with RPMI or with donor-matched plasma in 1.0 mL final vol. The cultures
were incubated for 18 h at 37°C and 5% CO2in loosely capped 12 ? 75 mm
snap-cap polypropylene tubes. After incubation, the separated plasma compo-
nents of the cultures were collected and frozen at –70°C until assayed.
Whole blood cultures with exogenous AAT
Whole blood from the same four healthy subjects was diluted 1:32 in RPMI
only, diluted 1:32 in RPMI with S. epi stimulation, or diluted 1:32 in RPMI
with S. epi stimulation in the presence of increasing concentrations of AAT or
albumin (1.0–8.0 mg/mL). AAT or albumin was added 1 h prior to S. epi
stimulation, and all final culture volumes were 1.0 mL in 12 ? 75 mm
snap-cap polypropylene tubes. After 18 h of incubation with loosely applied
caps, the separated plasma components of the blood cultures were aspirated
and frozen at –70°C until assayed.
2Journal of Leukocyte Biology
Volume 85, May 2009
Whole blood cultures with exogenous
Whole blood from each of four healthy subjects was diluted 1:16 in RPMI only,
diluted 1:16 in RPMI, and stimulated with S. epi or diluted 1:16 in RPMI and
stimulated with S. epi in the presence of 50 ?M AAPV-CMK or 0.1% DMSO
(vehicle control). AAPV-CMK or DMSO was added 1 h prior to S. epi
stimulation, and all final culture volumes were 1.0 mL in 12 ? 75 mm
snap-cap polypropylene tubes. After 18 h of incubation, the separated super-
natants of the cultured blood were aspirated and frozen at –70°C until assayed.
IL-8, IL-6, TNF-?, IL-1?, IL-1R antagonist (IL-1Ra), and IL-10 were mea-
sured using ECL assays, as described previously [1, 30–32]. All biotinylated
antibodies were obtained from eBioscience, Inc. (San Diego, CA, USA).
Antibodies obtained from R&D Systems (Minneapolis, MN, USA) were ruthe-
nylated using BV-TagTM-normal human serum-ester (Bioveris Corp., Gaithers-
burg, MD, USA). Cytokine measurements were performed using an M8 ECL
analyzer (Bioveris Corp.). The limit of detection for each cytokine ECL assay
was 10 pg/mL, and cytokine levels below the assay detection limit were
assigned the value 10 pg/mL.
The Mann-Whitney U test was used to compare plasma AAT concentrations
and whole blood cytokine production in AAT-deficient subjects and healthy
controls (see Table 1 and Fig. 1). For the whole blood dilution studies (see Fig.
2), cytokine concentrations were expressed using two methods: first, as directly
measured levels (see Fig. 2, A, C, E, and G) and second, as values calculated
by multiplication of the measured level by the dilution factor to obtain the
amount of cytokine produced per mL of whole blood that was in the cultures
(see Fig. 2, B, D, F, and H). For example, calculated levels in samples diluted
1:16 were obtained by multiplying the measured cytokine concentrations by
16. These calculations equalized the concentration of cytokine-producing
leukocytes in undiluted and diluted blood cultures in each donor. For studies
comparing cytokine levels in whole blood cultures in the absence or presence
of dilution, S. epi stimulation, or AAT (see Figs. 2, 3, and 5), differences
between experimental conditions were evaluated using repeated measures
ANOVA with Dunnett’s multiple comparison test. In the studies comparing
dilution with RPMI or plasma (see Fig. 4) and in the studies comparing dilution
alone with dilution and S. epi stimulation in the absence or presence of
AAPV-CMK (see Fig. 6), group means were compared using repeated mea-
sures ANOVA with Tukey’s multiple comparison test. P ? 0.05 was defined as
Cytokine production is increased in whole blood
from AAT-deficient patients
We compared cytokine production in stimulated whole blood
from nine subjects with genetic AAT deficiency to production
in 10 healthy controls. Table 1 depicts characteristics of the
two groups of participants. Blood from the AAT-deficient do-
nors was collected immediately prior to infusion of Prolastin?
or Zemaira?(clinical formulations of AAT) so that blood AAT
levels were at their respective nadirs. Plasma AAT concentra-
tions were significantly lower (P?0.004) in the AAT-deficient
patients compared with the healthy volunteers (median levels
of 1.67 mg/mL and 2.73 mg/mL, respectively). The blood for
these studies was cultured without dilution to maintain endog-
enous AAT concentrations. Whole blood from AAT-deficient
and control subjects was stimulated with S. epi and assessed for
cytokine production after 18 h of incubation. As shown in
Figure 1A, healthy controls produced a median 4.7 ng/mL
IL-8, whereas blood cultures in the AAT-deficient donors
produced a median 67.6 ng/mL (14.4 times the level in healthy
donors, P?0.0005). Median IL-6 and IL-1Ra production were
also increased significantly in the AAT-deficient group (3.4
and 8.4 times the median level in healthy controls, respec-
tively, Fig. 1, B and E). However, no significant difference in
median TNF-? or IL-1? production was observed in AAT-
deficient patients compared with controls (Fig. 1, C and D).
IL-10 levels were elevated in the AAT-deficient group (median
IL-10 in the AAT-deficient group was 1.8 times that of the
healthy controls), but this difference was not statistically sig-
nificant (Fig. 1F).
Diluting whole blood with RPMI increases
spontaneous cytokine production
To assess whether blood contains inhibitors of cytokine syn-
thesis, whole blood was collected from four healthy donors and
incubated for 18 h, undiluted or diluted with RPMI, to final
blood concentrations 1:4, 1:8, 1:16, or 1:32. Figure 2, A, C,
E, and G, shows spontaneous cytokine production presented as
measured concentrations. In Figure 2, B, D, F, and H, cytokine
levels are depicted after multiplication by the dilution factors
to obtain the calculated cytokine concentrations in the undi-
luted blood component of the cultures. These calculations were
designed to assess alterations in cytokine produced per white
blood cell in blood as a result of dilution. In Figure 2, A and
B, undiluted blood (dilution?0) produced 97.8 ? 56.4 pg/mL
IL-8 (mean?SEM). After multiplying by the dilution factor,
blood diluted to final concentrations 1:4, 1:8, 1:16, and 1:32
contained IL-8 levels of 93.9 ? 28.4 pg/mL, 118.9 ? 13.1
pg/mL, 192.7 ? 21.9 pg/mL, and 398.2 ? 26.1 pg/mL, re-
spectively (Fig. 2B). This represented a maximum mean 3.1-
fold increase in IL-8 levels in blood diluted 1:32 compared
with undiluted blood. In the same whole blood cultures, we
measured TNF-? (Fig. 2, C and D), IL-1? (Fig. 2, E and F),
and IL-1Ra (Fig. 2, G and H). As observed for IL-8, dilution of
blood with RPMI resulted in significant and dose-dependent
escalation in spontaneous TNF-?, IL-1?, and IL-1Ra after
adjustment for dilution. We observed maximum increases of
40-fold, 299-fold, and 18-fold in cytokine levels adjusted for
dilution compared with undiluted blood, respectively (Fig. 2,
D, F, and H). IL-6 and IL-10 concentrations in the same
TABLE 1. Characteristics of AAT-Deficient Subjects
and Healthy Controls
(n ? 9)
(n ? 10)
Age in yearsa
AAT phenotype: PiZZ
Plasma AAT concentration
aData shown as median (range).bDosing frequency ? 1 i.v. infusion per
week.cLevel determined immediately prior to AAT infusion; P ? 0.004.
ND ? Not determined. NA ? Not applicable.
Pott et al.
?-1-Antitrypsin reduces whole blood cytokines3
cultures were below the limit of assay detection for all condi-
tions tested (data not shown).
To determine if increased spontaneous cytokine production
was dependent on RPMI constituents, we repeated the blood
dilution experiments, except that blood was diluted in PBS or
HBSS instead of RPMI. In blood diluted 1:16 or 1:32 in PBS
or HBSS, IL-8, TNF-?, IL-1?, and IL-1Ra were increased to
similar extents, as observed for dilution in RPMI (data not
shown). These results indicate that cytokine increases were
dilution-dependent and not a result of RPMI components.
Dilution of blood augments S. epi-induced
We extended the dilution studies to assess the effect of dilution
on whole blood cultures stimulated with heat-killed S. epi,
which is a well-described cytokine inducer in vitro, and S.
epi-exposed blood reflects cytokine responses following activa-
tion of the TLR-2 . Blood was cultured undiluted, undi-
luted and stimulated with S. epi, or diluted and stimulated with
S. epi. As shown in Figure 3A, undiluted blood (dilution?0;
far-left bars) and undiluted blood stimulated with S. epi (dilu-
tion?0, second bars from left) produced 97.8 ? 56.4 pg/mL
and 4.2 ? 0.3 ng/mL IL-8 after 18 h of incubation, respec-
tively. Blood stimulated with S. epi and diluted 1:4, 1:8, 1:16,
or 1:32 with RPMI produced 88.6 ? 9.5 ng/mL, 163.7 ? 27.9
ng/mL, 233.6 ? 50.7 ng/mL, and 108.8 ? 18.1 ng/mL IL-8,
respectively. Diluting S. epi-stimulated blood 1:16 produced a
maximum mean 55-fold IL-8 increase (P?0.01) compared with
blood exposed to S. epi in the absence of dilution. In the same
whole blood cultures, we measured IL-6 (Fig. 3B), TNF-? (Fig.
3C), IL-1? (Fig. 3D), IL-1Ra (Fig. 3E), and IL-10 (Fig. 3F). As
observed for IL-8, cultures stimulated with S. epi and diluted
with RPMI demonstrated significant increases in cytokine con-
centrations compared with undiluted, S. epi-stimulated cul-
tures. Combined S. epi stimulation and RPMI dilution resulted
in maximum IL-6, TNF-?, IL-1?, IL-1Ra, and IL-10 levels
that were increased 1.3-fold (P?0.05), 8.1-fold (P?0.01),
1.3-fold (P?0.01), 5.6-fold (P?0.01), and 1.2-fold (P?not
significant) compared with levels observed with S. epi stimu-
lation alone (no dilution), respectively.
Effect of diluting blood with autologous plasma
on cytokine production
To determine if increased cytokine production in diluted
blood was a result of reduced concentration of inhibitory
substances in plasma, we examined the effect of blood
dilution with autologous plasma. Blood was collected from
four healthy donors and incubated for 18 h undiluted,
diluted with RPMI to a final blood concentration of 1:32, or
diluted 1:32 in autologous plasma. Cytokine levels were not
multiplied by the dilution factor (1:32) to directly compare
the cytokine levels in undiluted blood (dilution?0) and in
plasma-diluted blood. Compared with blood diluted in
RPMI, dilution in plasma suppressed TNF-?, IL-1?, and
IL-1Ra production (Fig. 4, B–D, respectively). IL-8 pro-
duction was decreased in RPMI and plasma-diluted samples
(Fig. 4A). Although IL-8 measured in the plasma-diluted
cultures was increased compared with the RPMI-diluted
cultures, the difference was not statistically significant. We
also measured IL-6 and IL-10 in these cultures, and levels
were below the detection limit (10 pg/mL) of the ECL assays
in all conditions tested (data not shown).
Fig. 1. Effect of AAT deficiency on cytokine production in stimulated whole blood. Cytokine production was measured in undiluted, S. epi-stimulated blood
obtained from nine AAT-deficient patients (●) and from 10 healthy controls (f). After 18 h of stimulation, supernatants were removed for cytokine assays, including
IL-8 (A), IL-6 (B), TNF-? (C), IL-1? (D), IL-1Ra (E), and IL-10 (F). Horizontal bars indicate median levels. **, P ? 0.005, and ***, P ? 0.0005, compared with
4 Journal of Leukocyte Biology
Volume 85, May 2009
Exogenous AAT inhibits cytokine production in
whole blood cultures
As AAT deficiency resulted in greater cytokine production in
whole blood cultures (Fig. 1), and other studies report anti-
inflammatory properties of AAT [7, 10, 11, 27, 34], we exam-
ined the effect of exogenous AAT on stimulated cytokine
production in whole blood. As shown in Figure 5A, whole
blood cultures diluted 1:32 with RPMI and stimulated with S.
epi (AAT?0) induced a mean 163.7 ? 30.9 ng/mL IL-8. The
addition of AAT reduced IL-8 in diluted and S. epi-stimulated
whole blood by a maximum of 99% using 8 mg/mL AAT
compared with cultures conducted in the absence of AAT. In
these cultures, we also measured IL-6 (Fig. 5B), TNF-? (Fig.
5C), IL-1? (Fig. 5D), IL-1Ra (Fig. 5E), and IL-10 (Fig. 5F).
Dose-dependent IL-6, TNF-?, and IL-1? suppression was
observed in the presence of AAT, with maximum mean reduc-
tions of 97%, 91%, and 47%, respectively, compared with
cultures without AAT, which did not affect the levels of stim-
ulated IL-1Ra and IL-10 significantly (Fig. 5, E and F).
To assess the specificity of AAT inhibition of stimulated
whole blood cytokine production, we used human serum-de-
rived albumin as a protein control. Whole blood was diluted
1:32 with RPMI and stimulated with S. epi in the absence
(control) or presence of 1–8 mg/mL albumin. In three separate
experiments, albumin did not affect diluted and S. epi-stimu-
lated whole blood production of any cytokine tested (IL-8, IL-6,
TNF-?, IL-1?, IL-1Ra, and IL-10; data not shown).
Effect of AAPV-CMK, a synthetic serine Pi, on
As AAT is the prototypical serine Pi in the circulation, we
surmised that AAT-induced suppression of whole blood proin-
flammatory cytokine production was a result of inhibition of
serine proteases. Therefore, we tested AAPV-CMK, a small-
molecule synthetic inhibitor of serine proteases in whole blood
cytokine production . Blood was collected from healthy
donors and diluted 1:16 with RPMI (control), diluted 1:16 with
RPMI and stimulated with S. epi, or diluted 1:16 with RPMI
and stimulated with S. epi in the presence of 50 ?M AAPV-
CMK (added 1 h prior to S. epi). After 18 h of incubation, the
mean IL-8 and IL-6 levels in blood diluted and stimulated with
S. epi were 163.8 ? 30.4 ng/mL and 33.7 ? 1.1 ng/mL,
Fig. 2. Spontaneous cytokine production in
whole blood diluted with RPMI. Heparinized
whole blood was incubated for 18 h undiluted
(dilution?0) or diluted in RPMI to the final
concentrations indicated on the horizontal
axes. After incubation, supernatants were re-
moved, and cytokine levels were quantified.
Cytokine levels are depicted as measured con-
centrations (A, C, E, and G) or multiplied by
the dilution factors (B, D, F, and H). Shown
are concentrations of IL-8 (A, B), TNF-?
(C, D), IL-1? (E, F), and IL-1Ra (G, H).
Cytokine concentrations are indicated on the vertical axes as means ? SEM in four separate donors. *, P ? 0.05; **, P ? 0.01, compared with dilu-
tion ? 0.
Pott et al.
?-1-Antitrypsin reduces whole blood cytokines5
respectively (Fig. 6, A and B, S. epi). Compared with diluted
and S. epi-stimulated blood, AAPV-CMK exposure resulted in
a statistically significant increase in IL-8 (234.3?12.4 ng/mL,
P?0.05) and IL-6 (45.0?3.5 ng/mL, P?0.05) production.
AAPV-CMK did not affect stimulated TNF-? (Fig. 6C) or
IL-1Ra significantly (Fig. 6E). In contrast, IL-1? was reduced
in diluted and S. epi-stimulated cultures containing AAPV-
CMK compared with diluted and S. epi-stimulated cultures
Fig. 3. Effect of whole blood dilution on S. epi-stimulated cytokine production. Heparinized whole blood was incubated for 18 h undiluted (dilution?0, far-left
bars), incubated undiluted with heat-killed S. epi as a stimulus (dilution?0, second bars from left), or with RPMI dilution to the levels indicated on the horizontal
axes and with S. epi stimulation. After incubation, cytokine concentrations were measured and expressed as concentration per mL of blood (multiplied by dilution
factor) for IL-8 (A), IL-6 (B), TNF-? (C), IL-1? (D), IL-1Ra (E), and IL-10 (F). Cytokine concentrations are indicated on the vertical axes as means ? SEM in four
separate donors. *, P ? 0.05, and **, P ? 0.01, compared with cultures stimulated with S. epi in the absence of dilution (dilution?0, second bars from left).
Fig. 4. Cytokine production in whole blood diluted with RPMI or autologous plasma. Whole blood was
incubated for 18 h undiluted (dilution?0), diluted 1:32 in RPMI [1:32 (RPMI)], or diluted 1:32 in
autologous plasma [1:32 (plasma)]. Supernatant cytokine concentrations are reported without multiplica-
tion by the dilution factor. Shown are levels of IL-8 (A), TNF-? (B), IL-1? (C), and IL-1Ra (D). Cytokine
concentrations are indicated on the vertical axes as means ? SEM in eight separate donors. For IL-8, **,
P ? 0.01, compared with dilution ? 0; for TNF-?, *, P ? 0.05, compared with dilution ? 0; for IL-1?,
*, P ? 0.05, compared with dilution ? 0 and to 1:32 (plasma); and for IL-1Ra, **, P ? 0.01, compared
with 1:32 (plasma).
6Journal of Leukocyte Biology
Volume 85, May 2009
(Fig. 6D) with a mean reduction of 62.5% compared with
blood-diluted 1:16 and stimulated with S. epi (P?0.01).
As AAPV-CMK was solubilized in DMSO, we examined the
DMSO effect in whole blood cytokine production. Whole blood
was diluted 1:16 with RPMI and S. epi, or blood was diluted 1:16
with S. epi and the equivalent volume of DMSO used in the
AAPV-CMK experiments (added 1 h prior to S. epi). After the whole
significantly affect diluted and S. epi-stimulated production of any
cytokine tested (IL-8, TNF-?, IL-1?, and IL-1Ra; data not shown).
Cytokine production in disease is studied commonly to eluci-
date pathogenesis or to quantify disease severity. Although
Fig. 5. Effect of exogenous AAT on cytokine production in diluted whole blood with S. epi stimulation. Whole blood was diluted 1:32 in RPMI and stimulated
with heat-killed S. epi in the absence (AAT?0) or presence of AAT added 1 h prior to S. epi. Final AAT concentrations are shown on the horizontal axes.
Supernatant cytokine concentrations were measured and expressed as concentrations per mL of blood (multiplied by dilution factor). Shown are levels of IL-8 (A),
IL-6 (B), TNF-? (C), IL-1? (D), IL-1Ra (E), and IL-10 (F). Cytokine concentrations are shown on the vertical axes as means ? SEM in cultures from four separate
donors. **, P ? 0.01, compared with AAT ? 0.
Fig. 6. Effect of the synthetic serine Pi AAPV-
CMK on cytokine production in diluted and S. epi-
stimulated whole blood, which was diluted 1:16 in
RPMI (Control), diluted 1:16 in RPMI and stimu-
lated with S. epi (S.epi), or diluted 1:16 in RPMI
and stimulated with S. epi in the presence of
50 ?M AAPV-CMK, added 1 h prior to S. epi
(S.epi?AAPV-CMK). After 18 h of incubation, su-
pernatant cytokine concentrations were determined
and expressed as concentration per mL of blood (multiplied by dilution factor). Shown are levels of IL-8 (A), IL-6 (B), TNF-? (C), IL-1? (D), and IL-1Ra (E).
Cytokine concentrations are shown on the vertical axes as means ? SEM in four separate donors. *, P ? 0.05; **, P ? 0.01; and ***, P ? 0.001, compared with
Pott et al.
?-1-Antitrypsin reduces whole blood cytokines7
cytokines are measured routinely in serum, circulating cyto-
kine levels are transient and reflect production, renal clear-
ance, hepatic metabolism, and binding to soluble cytokine
receptors or natural anticytokine antibodies [35–38]. Several
studies have shown that cytokine RNA expression in freshly
isolated whole blood is low or absent [39–41]. In the case of
IL-1?, low gene expression and absence of the IL-1? precursor
protein have been reported [40, 41]. In contrast, IL-18 gene
expression and the biologically inactive IL-18 precursor pro-
tein are present in the circulation of healthy individuals [41,
Although PBMC or monocyte-derived macrophages are com-
monly used to study in vitro cytokine production, the isolation
procedures are time-consuming and can result in nonspecific
stimulation of cytokine production . In addition, PBMC
populations do not reflect the ratios of cellular components in
circulating blood [3, 43]. For example, in PBMC preparations,
there are few or no neutrophils, and monocytes are three to five
times more abundant than in the circulation. For these reasons,
incubation of whole blood cultures for the analysis of cytokine
production and regulation emulates in vivo conditions more
closely. However, dilution of blood is necessary to maximize
cytokine synthesis in whole blood cultures [2–6], suggesting
that in vitro dilution of whole blood reduces the concentration
of factors present in the plasma that suppress cytokine pro-
duction. In the present study, we assessed the role of AAT in
cytokine production in whole blood cultures.
AAT deficiency is a genetic condition that increases the risk
of early onset and severe emphysema, chronic bronchitis,
bronchiectasis, and liver disease [7, 13, 15]. To examine AAT
anticytokine activity under in vivo-like conditions, we assessed
cytokine production in blood obtained from AAT-deficient
persons and from healthy controls. The whole blood was not
diluted to maintain endogenous AAT concentrations. S. epi-
stimulated blood collected from AAT-deficient individuals
demonstrated significantly greater IL-8, IL-6, and IL-1Ra pro-
duction compared with blood from healthy donors (Fig. 1).
These findings suggest that reduced AAT blood levels in
AAT-deficient subjects are associated with increased cytokine
production, implicating AAT as a cytokine-suppressive factor
in whole blood cultures.
These studies likely underestimate the cytokine-suppressive
effects of AAT. As our AAT-deficient patients received chronic
AAT replacement therapy, the difference in AAT levels be-
tween AAT-deficient patients and healthy controls was nar-
rowed. Despite AAT replacement therapy, the median AAT
level was reduced significantly in the AAT-deficient patients
(1.67 mg/mL) compared with healthy controls (2.73 mg/mL).
This difference was sufficient to result in significantly higher
IL-8, IL-6, and IL-1Ra production in the AAT-deficient group
(Fig. 1). It is possible that larger reductions in AAT (for
example, in AAT-deficient persons not receiving AAT supple-
mentation) would result in significantly enhanced levels of
other cytokines compared with healthy persons with normal
amounts of AAT.
When blood was diluted with RPMI tissue-culture medium,
spontaneous, proinflammatory cytokine production per mL of
blood was increased significantly (Fig. 2). Mean IL-8, TNF-?,
IL-1?, and IL-1Ra levels were increased 3.1-, 40-, 299-, and
18-fold compared with levels observed in undiluted blood.
Diluted whole blood cytokine levels that were not adjusted for
dilution demonstrated a dramatic reduction in IL-8, which was
not present for any other cytokine tested (Fig. 2A). In fact,
blood dilution resulted in increased, unadjusted TNF-? and
IL-1? concentrations, and IL-1Ra concentrations were only
slightly reduced. Although the reason for this anomalous IL-8
observation is uncertain, it may be relevant that of the cyto-
kines measured, only IL-8 is produced by polymorphonuclear
neutrophils (PMN). It is possible that blood dilution reduced
PMN concentrations in the cultures to the point that unad-
justed IL-8 levels declined precipitously. Alternatively, as
cell–cell contact between monocytes and T cells has been
shown to enhance IL-8 production , larger dilutions of
blood may limit the intercellular contact necessary for efficient
IL-8 production. It is noteworthy that dilution-induced dimi-
nution in monocyte concentrations did not result in similar
reductions in the monocyte-derived cytokines TNF-?, IL-1?,
and IL-1Ra (Fig. 2, C, E, and G). However, despite dilution-
induced reductions in unadjusted IL-8 levels, adjusted IL-8
concentrations (multiplication of IL-8 concentrations by the
dilution factor) increased with dilution (Fig. 2B).
We also examined the effect of dilution on cytokine produc-
tion in blood stimulated with S. epi (Fig. 3). Dilution-induced
increases in cytokine production were observed for each S.
epi-stimulated cytokine tested (IL-8, IL-6, TNF-?, IL-1?, IL-
1Ra, and IL-10). These results show that dilution increased
cytokine levels in stimulated whole blood beyond levels ob-
served with S. epi stimulation alone. In contrast to all other
measured cytokines, IL-8 production was not maximal at the
highest dilution but peaked at 1:16 dilution and decreased at
the 1:32 dilution (Fig. 3A). It is not clear why IL-8 production
did not continue to increase with higher dilution. It is possible
that in the presence of S. epi simulation and high (1:32)
dilution, the IL-8 contribution by PMN decreased, as described
for Figure 2A.
Three possibilities may explain increases in dilution-in-
duced cytokine production in the presence of S. epi stimulation
as observed in Figure 3: i) Dilution decreased cell–cell inter-
action, which may enhance cytokine production; ii) the con-
centration of cells was decreased in diluted samples, resulting
in an increase in the total amount of stimulus (S. epi) molecules
per cell; and iii) cytokine production was enhanced by reduc-
ing concentrations of natural inhibitors in the blood. As a
dilution-induced reduction in cell–cell interaction would be
expected to decrease the levels of cytokines produced , this
likely cannot explain the increase in cytokine production as-
sociated with dilution (Figs. 2 and 3). Furthermore, as the
fluid-phase concentration of S. epi was identical in each diluted
blood culture, the increased, total amount of S. epi per cell in
the cultures would not alter the magnitude of stimulation per
cell. Therefore, the hypothesis that soluble plasma inhibitors
are depleted by dilution is the most likely explanation.
To confirm the presence of cytokine inhibitors in the plasma
component of circulating blood, we diluted whole blood 1:32 in
RPMI or autologous plasma (Fig. 4). Dilution in plasma did not
increase levels of TNF-?, IL-1?, and IL-1Ra compared with
levels observed in undiluted blood. In no case did plasma
dilution increase cytokine synthesis significantly, as observed
8Journal of Leukocyte Biology
Volume 85, May 2009
for blood diluted in RPMI. These results support the contention
that dilution-induced cytokine synthesis is a result of reduced
concentrations of suppressive factors in plasma (as shown in
Fig. 2). Unlike the other cytokines we tested, IL-8 decreased in
response to dilution (Fig. 4A). As Figure 4 data are presented
as levels unadjusted for dilution, the results are similar to the
data shown in Figure 2A. As described in the text above that
refers to Figure 2, only IL-8 levels declined substantially with
dilution. We surmise this is a result of diminishing PMN
contribution to IL-8 production, which by each PMN, may not
respond to dilution with increased IL-8 synthesis to the extent
that the monocyte-derived cytokines increase with dilution.
Major protein components of plasma include albumin, Igs, ?
2-macroglobulin, and AAT. Several studies have suggested
that AAT possesses anti-inflammatory function [10, 23, 27, 34,
46–48], raising the possibility that AAT contributes to proin-
flammatory cytokine suppression in whole blood. As shown in
Figure 1, we demonstrated enhanced cytokine production in
stimulated cultures of whole blood obtained from AAT-defi-
cient patients compared with whole blood from healthy sub-
jects. These data identified AAT as a likely cytokine-suppres-
sive factor in blood. To determine if AAT inhibited cytokine
production directly in whole blood cultures, we added exoge-
nous AAT to whole blood that was diluted with RPMI and
stimulated with S. epi (Fig. 5). AAT (6–8 mg/mL) suppressed
IL-8, IL-6, TNF-?, and IL-1? production significantly by 99%,
97%, 91%, and 47%, respectively (Fig. 5, A–D). Relatively
high concentrations (6–8 mg/mL) of exogenous AAT were
required for substantial cytokine suppression in these experi-
ments. Although AAT levels of this magnitude can occur
during the acute-phase response, it is noteworthy that other
molecules in the circulation besides AAT suppress cytokines.
For example, ?
2-macroglobulin contributes to inflammatory response modu-
lation by binding and sequestering cytokines . Therefore,
significant cytokine suppression in diluted whole blood likely
requires high exogenous AAT concentrations to compensate for
the reduced levels of other (non-AAT) inhibitors. Also, high
AAT levels may have been necessary to overcome the large
cytokine induction effect provided by the combination of dilu-
tion and S. epi stimulation.
Unexpectedly, exogenous AAT at these same levels did not
suppress IL-1Ra and IL-10 significantly (Fig. 5, E and F), two
cytokines with anti-inflammatory activities. This suggests that
AAT preferentially inhibits proinflammatory cytokines, many
of which are regulated through the NF-?B pathway [50, 51],
and AAT has been shown to inhibit NF-?B activation [18, 46].
In contrast, IL-10 is stimulated through cAMP, and AAT has
been shown to increase cAMP synthesis and IL-10 production
in human monocytes in vitro [34, 52]. These opposing, AAT-
induced, intracellular signaling effects may explain why AAT
suppressed pro- but not anti-inflammatory cytokines in our in
We used a synthetic inhibitor of serine proteases, AAPV-
CMK, to determine if serine protease blockade is the mecha-
nism by which AAT suppresses cytokine production in whole
blood (Fig. 6).
AAPV-CMK (50 ?M) did not inhibit dilution and S. epi-
stimulated TNF-? or IL-1Ra production, suggesting that serine
protease blockade does not necessarily inhibit these cytokines
in whole blood. Interestingly, AAPV-CMK increased IL-8 and
IL-6 production to a significant extent, which contradicts the
hypothesis that serine protease inhibition is the mechanism by
which AAT suppressed cytokine production in these investi-
gations. Unlike the other cytokines assessed, IL-1? production
was inhibited significantly by AAPV-CMK added to stimulated
whole blood (Fig. 6D). Of the cytokines we tested, only IL-1?
is secreted following processing by the caspase-1 inflamma-
some . It is possible that inhibition of caspase-1 activity by
AAPV-CMK blocked IL-1? processing and prevented mature
IL-1? release into the culture supernatant. Collectively, these
AAPV-CMK results further suggest that serine protease inhi-
bition cannot completely account for AAT suppression of
proinflammatory cytokines, an observation noted by others [15,
These studies suggest that AAT is an endogenous inhibitor
of proinflammatory cytokine production in whole blood, and
AAT may participate in containing an aggressive innate im-
mune response to an inflammation-inducing stimulus. Further-
more, AAT activities separate from serine protease inhibition
likely participate in proinflammatory cytokine suppression.
Administration of exogenous AAT to patients with disease
characterized by excessive cytokine synthesis and inflamma-
tion may provide therapeutic benefit.
This work is supported by National Institutes of Health Grant
A115614 (to C. A. D.) and the Campbell Foundation (to L. S.).
The authors thank Dr. Kristin Morris and Scott Beard for
reviewing the manuscript and the AAT-deficient patients for
participating in these investigations.
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