In Vitro Incubation with Influenza VIrUS Primes Human Polymorphonuclear Leukocyte Generation of Superoxide William W. Busse, Rose F. Vrtis, Robert Steiner, and Elliot C. Dick Departments of Medicine and Preventive Medicine, University of Wisconsin Medical School, Madison, Wisconsin
Viral respiratory illnesses exacerbate asthma, increase airway responsiveness, and enhance the frequency of late asthmatic reactions. A number of mechanisms have been identified to explain how respiratory viral illnesses provoke wheezing, including enhanced inflammatory activity of leukocytes. To further understand how respiratory virus-caused illnesses promote leukocyte-dependent airway injury, the following study evaluated the effect of an in vitro incubation of influenza A virus on human polymorphonuclear leukocyte (PMN) generation of superoxide (02) . PMNs were isolated from anticoagulated human blood following density gradient centrifugation; purified PMNs were then incubated (37 0 C X 30 min) with influenza virus (PMN:virus ratio of5:1 [egg-infective dose 50%] and 10:1)in the presence of 10% autologous serum. After incubation, the viable PMNs (> 95% exclusion oftrypan blue) were activated, by the chemotactic peptide formyl-methionine-Ieucine-phenylalanine (tMLP) , calcium ionophore A23187, or phorbol myristate acetate (PMA) , and O2 generation was then measured. Generation of O2 to tMLP and A23187 was significantly enhanced from PMNs that had been incubated with influenza virus. Although influenza virus itself did not generate O2, it caused a transient increase in intracellular calcium ([Ca2+]i) when measured with Indo-l-Ioaded cells. These results suggest that influenza virus primes PMNs to generate increased amounts of O2 and that the priming effect is associated with a transient increase in [Ca"}. Consequently, we postulate that influenza virus priming produces PMN s of enhanced inflammatory potential to cause greater airway injury, obstruction, and responsiveness during a viral respiratory infection.
Viral respiratory illnesses frequently provoke airway hyperresponsiveness (1) and increase wheezing in some patients with asthma (2-5). Although the precise mechanisms by which viral respiratory illnesses alter airway function are unknown, considerable evidence suggests that the virus effects are multifactorial and include airway injury with sensitization of afferent vagus fibers (6), altered airway epithelial functions (7-9), diminished {j-adrenergic activity (10-13), production of virus-specific IgE antibody (14-16), and enhanced histamine release from human basophils (17, 18). Furthermore, we found that an experimentally induced viral respiratory illness enhanced airway responsiveness and
(Received in original form May 4, 1990 and in revised form September 26, 1990) Address correspondence to: William W. Busse, M.D., University of Wisconsin Hospital, H6/360 CSC, 600 Highland Avenue, Madison, WI 53792. Abbreviations: intracellular calcium, [Ca2+]i; cytochalasin B, CB; dimethyl sulfoxide, OMSO; ethylenediaminetetraacetic acid, EDTA; ethyleneglycol-bis-(I3-aminoethylether)-N,N-tetraacetic acid, EGTA; egginfective dose, EIDso; formyl-methionine-Ieucine-phenylalanine, tMLP; Hanks' balanced salt solution, HBSS; late asthmatic reaction, LAR; superoxide, 02"; platelet-activating factor, PAF; phorbol myristate acetate, PMA; polymorphonuclear leukocyte, PMN; red blood cell, RBC; superoxide dismutase, SOD. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 347-354, 1991
the frequency of late asthmatic reactions (LAR) (19). Because the airway obstruction that occurs during the LAR can be associated with increased airway responsiveness and recruitment of inflammatory cells such as neutrophils and eosinophils (20-24), it is felt to closely pattern the clinical features of chronic asthma (25-27). Although the precise mechanisms responsible for the induction of LAR during the viral respiratory illness were not established in these earlier studies (19), we hypothesized that viral augmentation of leukocyte function may be an important contributing factor. To analyze these relationships, we evaluated the in vitro effects of influenza virus on polymorphonuclear leukocyte (PMN) superoxide (02) generation.
Methods and Materials Human Subjects Venous blood was drawn from healthy, nonallergic adults after informed consent was obtained. All subjects were between 18 and 60 yr of age, had no history of asthma or allergic disease, and were taking no medication at the time of study. Reagents Hanks' balanced salt solution (HBSS) was obtained from GmCO (Grand Island, NY). Horse-heart ferricytochrome c (type VI), phorbol myristate (PMA) , calcium ionophore
348
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991
A23187, formyl-methionine-Ieucine-phenylalanine (fMLP) , cytochalasin B (CB), and superoxide dismutase (SOD) were purchased from Sigma Chemical Co. (St. Louis, MO). Indol/AM was purchased from Molecular Probes (Eugene, OR). Stock solutions of PMA (5 mg/ml), fMLP (10-2 M), and CB (10 mg/ml) were made in dimethyl sulfoxide (DMSO) and stored in aliquots at -80° C. Immediately before use, aliquots were thawed and adjusted to the appropriate concentrations with HBSS. DMSO did not affect cell viability or O2 generation at the concentrations used « 0.1%). Isolation of Neutrophil Suspensions Ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood (0.5 ml 2% EDTA/9.5 ml blood) was sedimented in 4.5% dextran in Tris-buffered saline solution, pH 7.4 (4 vol blood/l vol dextran for 45 min at 22° C). The plasma-leukocyte suspension was removed, layered over Ficoll-Hypaque (28), and then centrifuged (400 x g for 15 min at 22° C). After centrifugation, the mononuclear cell-rich band was removed, and the granulocyte-red blood cell (RBC) pellet resuspended in HBSS. Residual RBCs were lysed by hypotonic shock; the purified neutrophil (> 95 % pure) fractions were then washed in HBSS with 0.1% gelatin (HBSS-gel) and resuspended in HBSS. Cell viability was confirmed in all studies to be > 95 % as measured by trypan blue exclusion. 02" Generation Generation of O2 was measured as the SOD-inhibitable reduction of ferricytochrome c with a modified (29) microassay as previously described (30). With 96-well microtiter plates (lmmulon II; Dynatech,Alexandria, VA) and a 200-pJ reaction volume, 1 x 105 cells were added to 100 ILmollliter of cytochrome c in HBSS/gel. To initiate the reactions, the cells were incubated with selected activators (fMLP, calcium ionophore, or PMA). Immediately after the addition of an activator, the reaction wells were measured for absorbance at 550 nm in a Microplate Autoreader (EL 309; BioTek Instruments, Burlington, VT), followed by repeated readings for up to 1 h. Between absorbance measurements, the plates were placed in a 5 % CO2 incubator at 37° C. Each reaction was performed in duplicate and against an identical control reaction that contained 20 J.tg/ml of SOD. Results were adjusted to represent a l-ml reaction volume, and O2 generation was calculated with an extinction coefficient of 21.1 x 103 mol-liter? cm:' as nanomoles of cytochrome c reduced per 5 x lQ6 cells per time (minutes) minus SOD control. All incubations with activators and influenza took place in the presence of 10% autologous serum.
Indo-l Loading and Measurements of Intracellular Calcium ([Ca 2+]i) The synthesis of the calcium-sensitive fluorescent probes, such as Indo-l and its permeable, nonpolar ester derivative Indo-l/AM, provides an opportunity to measure the change in intracellular levels of free calcium in neutrophils following stimulation (31-32). Isolated PMNs (3 x lQ6/ml) were loaded with Indo-l/AM (in RPMI-0.5% bovine serum albumin) at a final concentration of 1 J.tM (33). After a 30-min incubation (37° C) in a light-excluding environment, the
PMN s were collected by centrifugation, washed twice, and placed in HBSS with 1 mM Ca2+. Control PMN s were treated the same, but without Indo-l. Fluorescent measurements were made in an SLM 8000™ C Photon Counting Spectrofluorometer (SLM Instruments, Urbana, IL) with an excitation wavelength of 355 and a ratio of emission wavelengths of 405 nm:485 nm. Calculations of [Ca"], were from the following equation as described by Grynkiewicz and associates (33):
where K; = 250 nM; R = fluorescence measure ratio of 405 nm:485 om; Rrnin = fluorescence ratio in the absence of Ca2+ after cell lysis; Rmax = fluorescence ratio in the presence of excess .Ca2+ after cell lysis; Sfi = fluorescence of the Indo-1 at 485 nm in the absence of Ca2+ after cell lysis; and Sb 2 = fluorescence of the Indo-l at 485 nm in the presence of excess Ca2+ after cell lysis. Influenza A Virus Incubation with PMNs Influenza A virus, an inhibitor-resistant recombinant of A/Eng/42/72 (H3N2 ) and A/PR8/34 (HoN1) , was grown in the allantoic sac of 9- to 10-d-old embryonate chick eggs (34). After incubation for 3 d at 35° C, followed by overnight incubation at 4 0 C, the allantoic fluid was harvested, clarified by centrifugation, and stored at -70 0 C until use. The virus infectivity was quantitated by the egg-infective dose 50 % (EID 5o) per milliliter. Allantoic fluid, which differed only in that it did not contain the virus particles, was added to a second aliquot of PMNs and served as a control. In all experiments, the isolated PMNs from the same subject were divided, with one-half being treated by influenza A virus and the other half by the allantoic fluid medium. Cell viability was maintained after a 30-min incubation with virus (trypan blue dye exclusion> 95 %). After incubation with either influenza A virus or the control medium, PMNs were centrifuged and resuspended in fresh calcium-containing HBSS, and 0; generation was measured as described above. Statistical Analysis The data were analyzed using a paired analysis by Student's t test, an analysis of variance, and/or univariate analysis. Differences between the influenza A virus-treated PMN s and controls were considered significant at P < 0.05.
Results Influenza A Virus Enhances PMN Generation of 02" When isolated human PMNs were incubated (370 C) with either noninfected allantoic fluid or live influenza A virus, cell viability (as measured by greater than 95 % exclusion of trypan blue dye) did not change nor was O2 generated (Figure 1). However, if the PMNs were then activated with the chemotactic stimuli fMLP (1 x 10-7 M), enhanced generation of 02" occurred with cells that had been incubated with influenza virus. The enhanced generation of 02" was most pronounced when the PMN:virus (EID 5o) ratio was 5:1 or 10:1;when the ratio ofPMN:virus in the incubation mixture
349
Busse, Vrtis, Steiner et al.: Influenza Virus Primes Neutrophils
21
12
..!!!
~IO
o
0 CD
:: 8 Q) a.
e
Q.
IN 0
• =Virus • =Placebo
Q)
a.
Q)
2
c
3
20
10
t
10
t
Virus
15
20
25
or
30
35
40
45
50
55
60
TIME (min)
CB
Figure 3. A comparison of O 2 generation to fMLP (l x 10- 7 M)
Placebo
Figure 1. A representative experiment comparing the effect of influenza virus (virus), or its non-virus-containing medium (placebo) on the generation of PMN O2 by tMLP (1 X 10- 7 M).
was increased to 1:1, O2 generation to tMLP was inhibited (data not shown). Thus, for subsequent experiments, a PMN:influenza A virus ratio of 5:1 was selected. With these established incubation and infectivity conditions, the effects of an in vitro virus incubation were examined on tMLP over a concentration range of 6 X 10-9 to 1 X 10-7 M (Figure 2). PMNs incubated with influenza virus generated significantly more O2 than did their matched controls. Furthermore, enhanced generation of O2 from influenza virus-treated PMNs was found throughout a 60min incubation period with 1 X 10-7 M tMLP (Figure 3). In additional studies, the effect of influenza A virus incubation on PMN O2 generation to calcium ionophore A23187 (Figure 4) and PMA was evaluated (Figure 5). In the presence of 5 J.tg/ml CB, calcium ionophore A23187 generation of PMN O2 was greater with cells incubated with influenza virus (data shown with A23187, 1.0 J.tM); a similar enhancement was noted with 0.1 J.tM (data not 20
shown). However, when PMA (3.0 ng/ml) was the activator, no enhancement in PMN O2 generation was noted from virus-treated PMNs; similarly, no enhancement of O2 generation occurred with other concentrations of.PMA, 0.3 and 1.0 nglml (data not shown). fMLP and A23187 activation of PMN O2 generation is associated with increased [Ca2+]j; in contrast, the PMN response to PMA is not associated with changes in [Ca'"], These data suggest that the enhancing effect of influenza virus on PMN generation of O2 is "calcium-dependent." Effect of Influenza A Virus on [Ca 2+J Concentrations To determine the effect of influenza virus on [Ca2+]j, Indol-loaded PMNs were incubated with virus and changes in fluorescence monitored (Figure 6, upper panel; Table 1). After the addition of influenza virus, PMN [Ca2+]j transiently rose; in contrast, no change in [Ca2+]j was noted with PMN s incubated with noninfected allantoic fluid (Figure 6, lower panel; Table 1). The addition of tMLP (1 X 10-8 M) caused a further incr~~ in PMN [Ca2+]j, which was 21
It)
~
Q;
o 15
0 0 0
,....
8ci
to
z
(620)
~3 z w
(407)
u
(/')
~2 o
(157)
::> ..J
u,
t
of.
o
5
10
15
20
25 30 35 40 TIME (min)
45
50
55
CB
Virus
O__=.'---+----i--+--+---+--+---+--+--.---_+_~ 60 0
Figure 5. A comparison of the effect of influenza virus (virus) and non-virus-containing medium (placebo) on PMN generation of 02" by 3.0 ng PMA. Values are mean of three experiments. Standard error bars are omitted for clarity.
5
(996)
4
TABLE 1
(/')
I-
The {Ca2+j i response to jMLP following incubation with either influenza A virus or non-virus-containing culture medium, with Indo-l-loaded PMNs* Baseline [Ca2+]i
Culture Medium
Influenza Virus
fMLP (1 X 10-8 M)
108 ± 13.0t
576 ± 30t 1,214 ± 182t
Z
::> w3
u Z w u
(/')
w2
50.0 ± 4.7 53.4 ± 4.9
58.3 ± 5.8
a::
o
:3u,
t
to
Definition ofabbreviations: [Ca2+li = intracellular calcium; fMLP = formylmethionine-leucine-phenylalanine; PMN s = polymorphonuclear leukocytes. * Values (in nanomoles) are mean ± SEM; n = 4. t p < 0.05 compared to respective baseline.
CB
Placebo
o Figure 6. Upperpanel: A representative experiment showing the effect of influenza virus (virus) on [Ca-"], and the subsequent response to tMLP (1 x 10- 8 M) with Indo-l-loaded PMNs. Lower panel: A representative experiment showing the effect of a nonvirus-containing medium (placebo) on [Ca2+]jand the subsequent response to fMLP (1 x 10- 8 M) with Indo-l-loaded PMNs.
greater in medium-treated cells (Table 1). Similar experiments were conducted in the presence of ethyleneglycol-bis({3-aminoethyl ether)-N,N -tetraacetic acid (EGTA) (7 mM) to chelate extracellular Ca2+. Although there was an initial decrease in PMN fluorescence after the addition of EGTA, the influenza virus still increased PMN [Ca2+]i (Table 2). Effect of Calcium Ionophore A23187 on PMN Generation of 02" to tMLP The above observations suggested that influenza A virus "primes" the PMN for activation by the chemotactic peptide fMLP, and this effect may be caused by a nonactivating rise in [Ca"], To simulate such conditions, PMNs were incubated with 0.01, 0.1, and 1.0 #LM concentrations of A23187
[ca2+)i : 9 4
in the absence of CB. Under these conditions, calcium ionophore did not generate 02" (Figure 7). However, when fMLP (1 x 10-8 M) was then added to the incubation mixture (in the presence of CB to parallel conditions of experiments with influenza virus), PMNs that had been incubated with either 0.1 or 1.0 ItM A23187 generated more O2 than the response to fMLP alone (Figure 7). Furthermore, when
TABLE 2
The PMN {Ca2+}; response to influenza A virus or non-virus-containing culture medium and then jMLP, in the presence of 7 mM EGTA with Indo-l-loaded PMNs* Baseline
55.0 ± 6.3 54.0 ± 5.3
EGTA
Culture Medium
21.6 ± 1.7 21.7 ± 1.4
23.0 ± 2.6
Influenza Virus
64.3
± 11.7t
fMLP (1 X 10-8 M)
559 ± 66.0t 315 ± 36.8U
Definition of abbreviations: [Ca 2+] ; = intracellular calcium; fMLP = fonnyl-methionine-Ieucine-phenylalanine; EGTA = ethyleneglycol-bis-(J3-aminoethyl ether)N,N'-tetraacetic acid. * Values (in nanomoles) are mean ± SEM; n = 3. t p < 0.05 compared to baseline. p < 0.05 compared to influenza virus.
*
Busse, Vrtis, Steiner et al.: Influenza Virus Primes Neutrophils
(n= 3, mean values)
12
351
5
(+CoI.I.O~Ml
•
en
~IO
4
:: 8 Ql a.
3
o ~
A=Control (660)
Ql
~ 6
e
2
Ql
a. ~ 4
[co+ 2 Ji
en Ql
"0
=87nM
t
E 2 c
Buffer
o
t
coror Buffer (OOI.O.I.1.0~M)
Figure 7. A comparison of the effect of a preincubation with 0.01, 0.1, or 1.0 JLM calcium ionophore A23187 (Cal) or buffer on the subsequent generation of O 2 from PMNs by fMLP (1 x 10- 7 M) in the presence of 5 JLg/ml cytochalasin B. Values are the mean ± SEM; n = 5. Standard error bars are omitted for clarity.
fluorescence was monitored with Indo-l-loaded cells, a [Ca2+]j increase occurred only in PMNs that had been incubated with either 0.1 or 1.0 ILM A23187, concentrations that enhanced FMLP generation of O 2 (Figure 8).
Discussion After an in vitro incubation with live influenza virus, human PMN s generated more 0; when subsequently activated by either the chemotactic peptide fMLP or calcium ionophore A23187. With the use of a fluorescent marker to monitor intracellular concentrations of Ca2+, we found influenza virus caused a transient increase in [Ca2+1 that was not associated with O2 generation. Furthermore, in a simulated model for this virus response, incubation of PMN s with selected concentrations of A23187 that did not generate O2 resulted in increased PMN [Ca2+];; however, when A23187treated cells were subsequently activated by fMLP, O2 generation was enhanced. Collectively, these data suggest that small, nonactivating (as measured by O2 generation) concentrations of influenza virus increased [Ca2+]; to prime the PMN and lead to an increased respiratory burst when subsequently exposed to an appropriate stimulus. The effect of respiratory viruses, particularly influenza, on PMN function has been of interest to many investigators, including ourselves. When Mills and colleagues (35) incubated influenza virus with PMN s, oxygen consumption, chemiluminescence, and the generation of O2 occurred. Although Mills and colleagues (35) performed their studies in the absence of serum, we also demonstrated that influenza virus elicited a PMN chemiluminescence response that was serum-dependent, proportional to the virus-antibody titer, and associated with consumption of complement during incubation with influenza virus (34, 36). In the present study, the concentrations of influenza virus that caused the enhanced response to fMLP did not, by themselves, stimulate O2 generation (Figure 1). The difference between our current observations and those previously reported (34, 36) suggest that chemiluminescence may be a more ..sensitive
5
en
B
~4
CalCium Ionophore A23186 OOlfLM
Z
:) 3
(506)
~
Z
L&J2 U
en
(122)
I L_~-----_-:---~
CB
Col (OOlfLMl
o
:)5 ..J
IJ..
t
t
L&J
a::
c- Calcium Ionophore A23186
4
0.1 fLM (719)
3
2
I
[CO+2)=91 nM
o
200
SECONDS Figure 8. One representative experiment comparing the effect of buffer (A = control) and two concentrations of calcium ionophore A23187 (B = 0.01 JLM, and C = 0.1 JLM) on PMN [Ca2+]; and the subsequent response to fMLP (1 x 10- 8 M).
method to detect initiation of the respiratory burst and, also, that the respiratory burst to influenza virus is atypical in that H20 2 (detected by chemiluminescence) is generated but O2 is not (37). Moreover, if PMNs were incubated with large concentrations of influenza, the subsequent response of the leukocyte to an activator was depressed and paralleled previous findings noted by us and others (35-38). Thus, the resultant effect of influenza virus on PMN function is dose dependent: large concentrations of virus suppress cell activity (34, 38, 39), while smaller, nonactivating inocula prime the cell to a subsequent stimulation by selected agonists. Mechanisms for the priming of neutrophils has considerable importance to host defense and inflammation. In previ-
352
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991
ously reported studies, Koenderman and co-workers (40) exposed isolated PMNs to subactivating concentrations of platelet-activating factor (PAF) or ionomycin; when these cells were subsequently activated by tMLP, granulocyte oxygen consumption was significantly enhanced. In these experiments, the priming effect of PAF and ionomycin was associated with a transient increase in PMN [Ca'"]; Depletion of PMN Ca2+ prevented priming by either PAF or ionomycin. Our observations also imply that priming of PMNs by influenza virus is associated with an increase in [Ca"']; Concentrations of influenza virus that prime the cell increase intracellular levels of Ca 2+ without causing PMN activation, as assessed by O2 generation; in contrast, priming did not occur when influenza virus did not increase PMN [Ca2+];. Thus, like PAF (40), the priming effect of influenza virus is associated with a transient increase in PMN [Ca2+];. Because the [Ca2+]i response to influenza virus also occurred when extracellular Ca2+ was chelated by EGTA, it is assumed that the transient increase in [Ca"], arises from intracellular sources rather than an extracellular influx. However, confirmation that shifts in neutrophil calcium levels are the cause of the influenza-enhanced response need further characterization. Our studies did not determine specifically how influenza virus influenced PMN [Ca'"]; Henricks and associates (41) found neuraminidase, from either influenza virus or Vibrio cholerae, partially removed PMN membrane sialic acid and enhanced O2 generation to opsonized Staphylococcus aureus; the investigators proposed that neuraminidase unmasks membrane receptor sites to increase the efficiency of the granulocyte's metabolic burst to particle activation. Furthermore, Langer and colleagues (42) described increased calcium exchangeability when neuraminidase removed sialic acid from cultured heart cells. Because we did not measure PMN membrane sialic acid after incubation with influenza virus, the contribution of neuraminidase to the enhanced response could not be determined. If the observations by Langer and colleagues (42) relate to the PMN, it is additional support for a link between the virus effect and altered [Ca2+]; concentrations. A variety of other biologically active compounds prime human neutrophils. VanEpps and, Garcia (43) exposed human PMNs to C5 fragments and found enhanced chemiluminescence upon subsequent stimulation with dissimilar soluble and particulate activators. Because influenza virus was incubated with fresh autologous serum, it is possible that C5 fragments were generated to cause, or contribute to, PMN priming (34). However, if C5 was formed in our experiments, it was not in concentrations sufficient to generate O2• Furthermore, because we did not determine specific antibody to the influenza virus in the sera, the pathway by which the virus interacts with the neutrophil might be quite different from subject to subject. If this is the case, the end result of an enhanced neutrophil response is similar as the relative increase in O 2 generation is comparable between individual subjects. Other processes may also participate in the priming process. For example, Weisbart and associates (44) and Zimmerli and co-workers (45) found increased receptors to the
chemotactic peptide tMLP on primed PMN s. Quantitation of tMLP receptors was not done in our study, but it is unlikely that increased membrane receptors could also explain the enhanced response to ionophore. Further, PMN priming may amplify stimulus-response coupling from the receptor to the measured cellular action; Kuhns and colleagues (46) found ATP-induced enhancement of O2 generation was associated with a shortened lag time in the neutrophil response to tMLP without a change in the median effective dose for the chemotactic factor. These observations suggest that signal transduction, not altered receptor affinity, can cause enhanced oxidative activity. Parallel to our observations, Kuhns and colleagues (46) found that ATP-induced priming was associated with a rise in PMN [Ca2+]; without cell activation. Finally, Hughes and co-workers (47) and Guthrie and associates (48) suggest that either increased NADPH oxidase levels, or its activity, contributes to neutrophil priming. The effects of influenza virus on NADPH oxidase and other enzymes were not determined. Our observation that respiratory viruses enhance PMN phlogistic activity complements reports demonstrating that these microorganisms promote selected inflammatory responses. When human leukocytes are incubated with respiratory viruses, IgE-dependent basophil histamine release increases, an effect mediated, in part, by the generation of interferon (17, 18). Enhanced basophil histamine release also occurs during in vivo viral respiratory infections (19) to corroborate the potential validity of the in vitro modeling of this response. Further, if human leukocytes are incubated with respiratory viruses, basophils, but not other leukocytes, have greater chemotactic response to complement particles and lymphocyte-derived proteins (49). Finally, viral respiratory infections (11) and invitro incubations with viruses (12, 13) depress {j-adrenergic regulation of lysosomal enzyme release, a consequence that translates to promotion of PMNdriven inflammation. Although it remains to be determined precisely how, and if, toxic oxygen radicals contribute to airways injury in asthma, there is sufficient evidence that these products are injurious to the lung in other pulmonary diseases (50, 51). Our in vitro studies indicate yet another mechanism by which respiratory viruses can compound their ability to injure the airway either directly or by generation of more toxic oxygen products. Finally, although the significance of these in vitro observations to virus-provoked asthma has yet to be fully established, they suggest the following clinical possibility and relationship to the development of late asthmatic reactions (Figure 9). During viral respiratory illnesses, neutrophils become primed. Consequently, recruitment of leukocytes to the airways during allergic reactions will include primed neutrophils that have a greater inflammatory potential. When the primed PMN is activated (possibly by substances such as leukotriene B4) , the resultant bronchial inflammation is greater than nonviral respiratory illness periods and hence the probability for late asthmatic reactions increased. Because similarities have already been found between in vivo and in vitro virus effects on leukocyte function (11, 12, 18, 19), we feel that the proposed events suggested in Figure 9 are a possibility. However, these studies were conducted with
Busse, Vrtis, Steiner et al.: Influenza Virus Primes Neutrophils
The Effect of Respiratory Viruses on PMN Function
PMN~ --\.d
+ Respiratory Virus
Activation
© ~
~ca ~
c onsequence on Airways Inflammation Hyperresponsiviness Obstruction
Figure 9. A hypothetical scheme showing the possible effect of a respiratory virus on PMN function. It is postulated that the respiratory virus primes the PMN to release more 02" when activated. If activation of the primed PMN occurs in the airway, the consequence is greater inflammation, obstruction, and hyperresponsiveness.
leukocytes from normal individuals; whether similar responses will be noted with neutrophils from allergic or asthma subjects will be of considerable interest and relevance to the.issue of virus-induced reactive airways disease. Future studies, therefore, will be needed to establish this possibility and the precise mechanism by which respiratory viruses prime the neutrophil. Acknowledgments: This work was supported by Grants AI-15685, AI-23181, AI-I0404, and HL-44098 from the National Institutes of Health.
References 1. Frick, W. E., and W. W. Busse. 1988. Respiratory infections: their role in airway responsiveness and pathogenesis of asthma. Clin. Chest Med. 9:539-549. 2. McIntosh, K., E. F. Ellis, L. S. Hoffman, T. G. Lybass, J. J. Eller, and V. A. Fulginiti. 1973. The association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children. l. Pediatr. 82:578-590. 3. Minor, T. E., E. C. Dick, A. N. DeMeo, J. J. Ouellette, M. Cohen, and C. E. Reed. 1974. Viruses as precipitants of asthmatic attacks in children. lAMA 227:292-298. 4. Minor, T. E., E. C. Dick, J. W. Baker, J. J. Ouellette, M. Cohen, and C. E. Reed. 1976. Rhinovirus and influenza type A infections as precipitants of asthma. Am. Rev. Respir. Dis. 113:149-153. 5. Hudgel, D. W., C. Langston, Jr., J. C. Selner, and K. McIntosh. 1979. Viral and bacterial infections in adults with chronic asthma. Am. Rev. Respir. Dis. 120:393-397. 6. Empey, D. W., L. A. Laitinen, L. Jacobs, W. M. Gold, andJ. A. Nadel. 1976. Mechanisms of bronchial hyperreactivity in normal subjects after upper respiratory tract infection. Am. Rev. Respir. Dis. 113:131-139. 7. Saban, R., E. C. Dick, R. I. Fishleder, and C. K. Buckner. 1987. Enhancement by parainfluenza 3 infection of contractile responses to substance P and capsaicin in airway smooth muscle from the guinea pig. Am. Rev. Respir. Dis. 136:586-591. 8. Jacoby, D. B., J. Tamaoki, D. B. Borson, andJ. A. Nadel. 1988. Influenza infection causes airway hyperresponsiveness by decreasing enkephalinase. J. Appl. Physiol. 64:2653-2658. 9. Dusser, D. J., D. B. Jacoby, T. D. Djokic, I. Rubinstein, D. B. Borson, and J. A. Nadel. 1989. Virus induces airway hyperresponsiveness to tachykinins: role of neutral endopeptidase. J. Appl. Physiol. 67: 1504-1511. 10. Szentivanyi, A. 1968. The beta-adrenergic theory ofthe atopic abnormality in asthma. J. Allergy 42:203-223. 11. Busse, W. W. 1977. Decreased granulocyte response to isoproterenol in asthma during upper respiratory infections. Am. Rev. Respir. Dis. 115:783-791.
353
12. Busse, W. W., W. Cooper, D. M. Warshauer, E. C. Dick, I. H. L. Wallow, and R. Albrecht. 1979. Impairment of isoproterenol, H2 histamine, and prostaglandin E 1 response of human granulocytes after incubation in vitro with live influenza vaccines. Am. Rev. Respir. Dis. 119:561-569. 13. Busse, W. W., C. L. Anderson, E. C. Dick, and D. Warshauer. 1980. Reduced granulocyte response to isoproterenol, histamine, and prostaglandin E 1 after in vitro incubation with rhinovirus 16. Am. Rev. Respir. Dis. 122:641-646. 14. Welliver, R. C., D. T. Wong, M. Sun, E. Middleton, Jr., R. S. Vaughan, and P. L. Ogra. 1981. The development of respiratory syncytial virus-specific IgE and the release of histamine in nasopharyngeal secretions after infection. N. Engl. J. Med. 305:841-846. 15. Welliver, R. C., D. T. Wong, E. Middleton, Jr., M. Sun, N. McCarthy, and P. L. Ogra. 1982. Role of parainfluenza virus-specific IgE in pathogenesis of croup and wheezing subsequent to infection. J. Pediatr. 101:889-896. 16. Welliver, R. L., M. Sun, D. Rinaldo, and P. L. Ogra. 1986. Predictive value of respiratory syncytial virus-specific IgE responses for recurrent wheezing following bronchiolitis. J. Pediatr. 109:776-780. 17. Ida, S., J. J. Hooks, R. P. Siranganian, and A. L. Notkins. 1977. Enhancement of IgE-mediated histamine release from human basophils by viruses. Role of interferon. J. Exp. Med. 145:892-896. 18. Busse, W. W., C. A. Swenson, E. C. Borden, M. W. Treuhaft, and E. C. Dick. 1983. Effect of influenza A virus on leukocyte histamine release. J. Allergy Clin. Immunol. 71:382-388. . 19. Lemanske, R. F., Jr., E. C. Dick, C. A. Swenson, R. F. Vrtis, and W. W. Busse. 1989. Rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions. J. Clin. Invest. 83: 1-10. 20. deMonchy, J. G. R., H. F. Kauffman, P. Venge etal. 1985. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions. Am. Rev. Respir. Dis. 131:373-376. 21. Metzger, W. J., H. B. Richerson, K. Worden, M. Monick, and G. W. Hunninghake. 1986. Bronchoalveolar lavage of allergic asthmatic patients following allergen provocation. Chest 89:477-483. 22. Carroll, M. P., S. R. Durham, G. Walsh, andA. B. Kay. 1985. Activation of neutrophils and monocytes after allergen- and histamine-induced bronchospasm. J. Allergy Clin. Immunol. 75:290-296. 23. Murphy, K. R., M. C. Wilson, C. G. Irvin et al. 1986. The requirement for polymorphonuclear leukocytes in the late asthmatic response and heightened airways reactivity in an animal model. Am. Rev. Respir. Dis. 134:62-68. 24. Fabbri, L. M., P. Boschetto, E. Zocca et al. 1987. Bronchoalveolar neutrophilia during late asthmatic reactions induced by toluene diisocyanate. Am. Rev. Respir. Dis. 136:36-42. 25. Metzger, W. J., G. W. Hunninghake, and H. B. Richerson. 1985. Late asthmatic responses: injury inquiry into mechanisms and significance. Clin. Rev. Allergy 3:145-165. 26. O'Byrne, P. M., J. Dolovich, and F. E. Hargreave. 1987. Late asthmatic responses. Am. Rev. Respir. Dis. 136:740-751. 27. Lemanske, R. R., Jr., and M. A. Kaliner. 1988. Late-phase allergic reactions. In Allergy: Principles and Practice. E. Middleton, Jr., C. E. Reed, E. F. Ellis, N. F. Adkinson, Jr., and J. W. Yanginger, editors. C. V. Mosby, St. Louis, MO. 224-246. 28. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21(Suppl. 97):77-89. 29. Sedgwick, J. B., R. F. Vrtis~/M. F. Gourley, and W. W. Busse. 1988. Stimulus-dependent differences in superoxide anion generation by normal eosinophils and neutrophils. J. Allergy Clin. Immunol. 81:876-883. 30. Pick, E., and D. Mizel. 1981. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. J. Immunol. Methods 46:211-226. 31. Lazzari, K. G., P. S. Proto, and E. R. Simons. 1986. Simultaneous measurement of stimulus-induced changes in cytoplasmic Ca2+ and in membrane potential of human neutrophils. J. Bioi. Chem. 261:9710-9713. 32. Nasmith, P. E., and S. Grinstein. 1987. Are Ca 2+ channels in neutrophils activated by a rise in cytosolic Ca2+? FEBS Lett. 221:95-100. 33. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. A new generation of Ca 2+ indicators with greatly improved fluorescence properties. J. BioI. Chem. 360:3440-3450. 34. Shult, P. A., E. C. Dick, K. A. Joiner, and W. W. Busse. 1985. Role of serum in stimulation of polymorphonuclear leukocyte, luminol-dependent chemiluminescence by influenza A. Am. Rev. Respir. Dis. 131:267-272. 35. Mills, E. L., Y. Debets-Ossenkopp, H. A. Verbrugh, and J. Verhoef. 1981. Initiation of the respiratory burst of human neutrophils by influenza virus. Infect. Immun. 32:1200-1205. 36. Busse, W. W., andJ. M. Sosman. 1981. Altered luminol-dependent granulocyte chemiluminescence during an in vitro incubation with influenza vaccine. Am. Rev. Respir. Dis. 123:654-658. 37. Hartshorn, K. C., M. Collamer, M. R. White, J. H. Schwartz, and A. I. Tauber. 1990. Characterization of influenza A virus activation of the human neutrophil. Blood 75:218-226. 38. Faden, H., P. Sutyla, and P. Ogra. 1979. Effect of viruses on luminol-
354
39.
40. 41.
42. 43. 44.
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991
dependent chemiluminescence of human neutrophils. Infect. Immun. 24:673-678. Cassidy, L. F., D. S. Lyles, and J. S. Abramson. 1989. Depression of polymorphonuclear leukocyte functions by purified influenza virus hemagglutinin and sialic acid-binding lectins. J. Immunol. 142:44014406. Koenderman, L., Yazdanbakhsh, D. Roos, and A. J. Verhoeven. 1989. Dual mechanisms in priming of the chemoattractant-induced respiratory burst in human granulocytes. J. Immunol. 142:623-628. Henricks, P. A. J., M. E. van Erne-van der Tol, and J. Verhoef. 1982. Partial removal of sialic acid enhances phagocytosis and the generation of superoxide and chemiluminescence by polymorphonuclear leukocytes. J. Immunol. 129:745-750. Langer, G. A., J. S. Frank, L. M. Nudd, and K. Seraydavin. 1976. Sialic acid: effect of removal on calcium exchangeability of cultured heart cells. Science 193:1013-1015. VanEpps, D. E., andM. L. Garcia. 1981. Enhancement of'neutrophil function as a result of prior exposure to chemotactic factor. J. Clin. Invest. 66:167-175. Weisbart, R. H., D. W. Golde, and J. C. Basson. 1986. Biosynthetic human GM-CSF modulates the number and affinity of neutrophil f-met-leuphe receptors. J. Immunol. 137:3584-3587.
45. Zimmerli, W., B. E. Seligmann, and 1. I. Gallin. 1987. Neutrophils are hyper-polarized after exudation and show an increased depolarization response to formyl-peptide but not to phorbol myristate acetate. Eur. J. Clin. Invest. 17:435-441. 46. Kuhns, D. B., D. G. Wright, J. Nath, S. S. Kaplan, and R. E. Basford. 1988. ATP induces transient elevations of [Ca2+]i in human neutrophils and primes those cells for enhanced 02" generation. Lab. Invest. 58:448-453. 47. Hughes, V., J. M. Humphreys, and S. W. Edwards. 1987. Protein synthesis is activated in primed neutrophils: a possible role in inflammation. Biosci. Rep. 7:881-890. 48. Guthrie, L. A., L. C. McPhail, P. M. Henson, and R. B. Johnston, Jr. 1984. Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide. J. Exp. Med. 160:1656-1671. 49. Lett-Brown, M. A., M. Aelvolet, J. J. Hooks, J. A. Georgiades, D. O. Thueson, and J. A. Grant. 1981. Enhancement of basophil chemotaxis in vitro by virus-induced interferon. J. Clin. Invest. 67:547-552. 50. Fantone, J. C., D. E. Feltner, J. K. Brieland, and P. A. Ward. 1987. Phagocyte cell-derived inflammatory mediators and lung disease. Chest 91:428-435. 51. Heffner, J. E., and J. E. Repine. 1989. Pulmonary strategies of antioxidant defense. Am. Rev. Respir. Dis. 140:531-554.
Viral respiratory illnesses exacerbate asthma, increase airway responsiveness, and enhance the frequency of late asthmatic reactions. A number of mechanisms have been identified to explain how respiratory viral illnesses provoke wheezing, including enhanced inflammatory activity of leukocytes. To further understand how respiratory virus-caused illnesses promote leukocyte-dependent airway injury, the following study evaluated the effect of an in vitro incubation of influenza A virus on human polymorphonuclear leukocyte (PMN) generation of superoxide (02) . PMNs were isolated from anticoagulated human blood following density gradient centrifugation; purified PMNs were then incubated (37 0 C X 30 min) with influenza virus (PMN:virus ratio of5:1 [egg-infective dose 50%] and 10:1)in the presence of 10% autologous serum. After incubation, the viable PMNs (> 95% exclusion oftrypan blue) were activated, by the chemotactic peptide formyl-methionine-Ieucine-phenylalanine (tMLP) , calcium ionophore A23187, or phorbol myristate acetate (PMA) , and O2 generation was then measured. Generation of O2 to tMLP and A23187 was significantly enhanced from PMNs that had been incubated with influenza virus. Although influenza virus itself did not generate O2, it caused a transient increase in intracellular calcium ([Ca2+]i) when measured with Indo-l-Ioaded cells. These results suggest that influenza virus primes PMNs to generate increased amounts of O2 and that the priming effect is associated with a transient increase in [Ca"}. Consequently, we postulate that influenza virus priming produces PMN s of enhanced inflammatory potential to cause greater airway injury, obstruction, and responsiveness during a viral respiratory infection.
Viral respiratory illnesses frequently provoke airway hyperresponsiveness (1) and increase wheezing in some patients with asthma (2-5). Although the precise mechanisms by which viral respiratory illnesses alter airway function are unknown, considerable evidence suggests that the virus effects are multifactorial and include airway injury with sensitization of afferent vagus fibers (6), altered airway epithelial functions (7-9), diminished {j-adrenergic activity (10-13), production of virus-specific IgE antibody (14-16), and enhanced histamine release from human basophils (17, 18). Furthermore, we found that an experimentally induced viral respiratory illness enhanced airway responsiveness and
(Received in original form May 4, 1990 and in revised form September 26, 1990) Address correspondence to: William W. Busse, M.D., University of Wisconsin Hospital, H6/360 CSC, 600 Highland Avenue, Madison, WI 53792. Abbreviations: intracellular calcium, [Ca2+]i; cytochalasin B, CB; dimethyl sulfoxide, OMSO; ethylenediaminetetraacetic acid, EDTA; ethyleneglycol-bis-(I3-aminoethylether)-N,N-tetraacetic acid, EGTA; egginfective dose, EIDso; formyl-methionine-Ieucine-phenylalanine, tMLP; Hanks' balanced salt solution, HBSS; late asthmatic reaction, LAR; superoxide, 02"; platelet-activating factor, PAF; phorbol myristate acetate, PMA; polymorphonuclear leukocyte, PMN; red blood cell, RBC; superoxide dismutase, SOD. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 347-354, 1991
the frequency of late asthmatic reactions (LAR) (19). Because the airway obstruction that occurs during the LAR can be associated with increased airway responsiveness and recruitment of inflammatory cells such as neutrophils and eosinophils (20-24), it is felt to closely pattern the clinical features of chronic asthma (25-27). Although the precise mechanisms responsible for the induction of LAR during the viral respiratory illness were not established in these earlier studies (19), we hypothesized that viral augmentation of leukocyte function may be an important contributing factor. To analyze these relationships, we evaluated the in vitro effects of influenza virus on polymorphonuclear leukocyte (PMN) superoxide (02) generation.
Methods and Materials Human Subjects Venous blood was drawn from healthy, nonallergic adults after informed consent was obtained. All subjects were between 18 and 60 yr of age, had no history of asthma or allergic disease, and were taking no medication at the time of study. Reagents Hanks' balanced salt solution (HBSS) was obtained from GmCO (Grand Island, NY). Horse-heart ferricytochrome c (type VI), phorbol myristate (PMA) , calcium ionophore
348
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991
A23187, formyl-methionine-Ieucine-phenylalanine (fMLP) , cytochalasin B (CB), and superoxide dismutase (SOD) were purchased from Sigma Chemical Co. (St. Louis, MO). Indol/AM was purchased from Molecular Probes (Eugene, OR). Stock solutions of PMA (5 mg/ml), fMLP (10-2 M), and CB (10 mg/ml) were made in dimethyl sulfoxide (DMSO) and stored in aliquots at -80° C. Immediately before use, aliquots were thawed and adjusted to the appropriate concentrations with HBSS. DMSO did not affect cell viability or O2 generation at the concentrations used « 0.1%). Isolation of Neutrophil Suspensions Ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood (0.5 ml 2% EDTA/9.5 ml blood) was sedimented in 4.5% dextran in Tris-buffered saline solution, pH 7.4 (4 vol blood/l vol dextran for 45 min at 22° C). The plasma-leukocyte suspension was removed, layered over Ficoll-Hypaque (28), and then centrifuged (400 x g for 15 min at 22° C). After centrifugation, the mononuclear cell-rich band was removed, and the granulocyte-red blood cell (RBC) pellet resuspended in HBSS. Residual RBCs were lysed by hypotonic shock; the purified neutrophil (> 95 % pure) fractions were then washed in HBSS with 0.1% gelatin (HBSS-gel) and resuspended in HBSS. Cell viability was confirmed in all studies to be > 95 % as measured by trypan blue exclusion. 02" Generation Generation of O2 was measured as the SOD-inhibitable reduction of ferricytochrome c with a modified (29) microassay as previously described (30). With 96-well microtiter plates (lmmulon II; Dynatech,Alexandria, VA) and a 200-pJ reaction volume, 1 x 105 cells were added to 100 ILmollliter of cytochrome c in HBSS/gel. To initiate the reactions, the cells were incubated with selected activators (fMLP, calcium ionophore, or PMA). Immediately after the addition of an activator, the reaction wells were measured for absorbance at 550 nm in a Microplate Autoreader (EL 309; BioTek Instruments, Burlington, VT), followed by repeated readings for up to 1 h. Between absorbance measurements, the plates were placed in a 5 % CO2 incubator at 37° C. Each reaction was performed in duplicate and against an identical control reaction that contained 20 J.tg/ml of SOD. Results were adjusted to represent a l-ml reaction volume, and O2 generation was calculated with an extinction coefficient of 21.1 x 103 mol-liter? cm:' as nanomoles of cytochrome c reduced per 5 x lQ6 cells per time (minutes) minus SOD control. All incubations with activators and influenza took place in the presence of 10% autologous serum.
Indo-l Loading and Measurements of Intracellular Calcium ([Ca 2+]i) The synthesis of the calcium-sensitive fluorescent probes, such as Indo-l and its permeable, nonpolar ester derivative Indo-l/AM, provides an opportunity to measure the change in intracellular levels of free calcium in neutrophils following stimulation (31-32). Isolated PMNs (3 x lQ6/ml) were loaded with Indo-l/AM (in RPMI-0.5% bovine serum albumin) at a final concentration of 1 J.tM (33). After a 30-min incubation (37° C) in a light-excluding environment, the
PMN s were collected by centrifugation, washed twice, and placed in HBSS with 1 mM Ca2+. Control PMN s were treated the same, but without Indo-l. Fluorescent measurements were made in an SLM 8000™ C Photon Counting Spectrofluorometer (SLM Instruments, Urbana, IL) with an excitation wavelength of 355 and a ratio of emission wavelengths of 405 nm:485 nm. Calculations of [Ca"], were from the following equation as described by Grynkiewicz and associates (33):
where K; = 250 nM; R = fluorescence measure ratio of 405 nm:485 om; Rrnin = fluorescence ratio in the absence of Ca2+ after cell lysis; Rmax = fluorescence ratio in the presence of excess .Ca2+ after cell lysis; Sfi = fluorescence of the Indo-1 at 485 nm in the absence of Ca2+ after cell lysis; and Sb 2 = fluorescence of the Indo-l at 485 nm in the presence of excess Ca2+ after cell lysis. Influenza A Virus Incubation with PMNs Influenza A virus, an inhibitor-resistant recombinant of A/Eng/42/72 (H3N2 ) and A/PR8/34 (HoN1) , was grown in the allantoic sac of 9- to 10-d-old embryonate chick eggs (34). After incubation for 3 d at 35° C, followed by overnight incubation at 4 0 C, the allantoic fluid was harvested, clarified by centrifugation, and stored at -70 0 C until use. The virus infectivity was quantitated by the egg-infective dose 50 % (EID 5o) per milliliter. Allantoic fluid, which differed only in that it did not contain the virus particles, was added to a second aliquot of PMNs and served as a control. In all experiments, the isolated PMNs from the same subject were divided, with one-half being treated by influenza A virus and the other half by the allantoic fluid medium. Cell viability was maintained after a 30-min incubation with virus (trypan blue dye exclusion> 95 %). After incubation with either influenza A virus or the control medium, PMNs were centrifuged and resuspended in fresh calcium-containing HBSS, and 0; generation was measured as described above. Statistical Analysis The data were analyzed using a paired analysis by Student's t test, an analysis of variance, and/or univariate analysis. Differences between the influenza A virus-treated PMN s and controls were considered significant at P < 0.05.
Results Influenza A Virus Enhances PMN Generation of 02" When isolated human PMNs were incubated (370 C) with either noninfected allantoic fluid or live influenza A virus, cell viability (as measured by greater than 95 % exclusion of trypan blue dye) did not change nor was O2 generated (Figure 1). However, if the PMNs were then activated with the chemotactic stimuli fMLP (1 x 10-7 M), enhanced generation of 02" occurred with cells that had been incubated with influenza virus. The enhanced generation of 02" was most pronounced when the PMN:virus (EID 5o) ratio was 5:1 or 10:1;when the ratio ofPMN:virus in the incubation mixture
349
Busse, Vrtis, Steiner et al.: Influenza Virus Primes Neutrophils
21
12
..!!!
~IO
o
0 CD
:: 8 Q) a.
e
Q.
IN 0
• =Virus • =Placebo
Q)
a.
Q)
2
c
3
20
10
t
10
t
Virus
15
20
25
or
30
35
40
45
50
55
60
TIME (min)
CB
Figure 3. A comparison of O 2 generation to fMLP (l x 10- 7 M)
Placebo
Figure 1. A representative experiment comparing the effect of influenza virus (virus), or its non-virus-containing medium (placebo) on the generation of PMN O2 by tMLP (1 X 10- 7 M).
was increased to 1:1, O2 generation to tMLP was inhibited (data not shown). Thus, for subsequent experiments, a PMN:influenza A virus ratio of 5:1 was selected. With these established incubation and infectivity conditions, the effects of an in vitro virus incubation were examined on tMLP over a concentration range of 6 X 10-9 to 1 X 10-7 M (Figure 2). PMNs incubated with influenza virus generated significantly more O2 than did their matched controls. Furthermore, enhanced generation of O2 from influenza virus-treated PMNs was found throughout a 60min incubation period with 1 X 10-7 M tMLP (Figure 3). In additional studies, the effect of influenza A virus incubation on PMN O2 generation to calcium ionophore A23187 (Figure 4) and PMA was evaluated (Figure 5). In the presence of 5 J.tg/ml CB, calcium ionophore A23187 generation of PMN O2 was greater with cells incubated with influenza virus (data shown with A23187, 1.0 J.tM); a similar enhancement was noted with 0.1 J.tM (data not 20
shown). However, when PMA (3.0 ng/ml) was the activator, no enhancement in PMN O2 generation was noted from virus-treated PMNs; similarly, no enhancement of O2 generation occurred with other concentrations of.PMA, 0.3 and 1.0 nglml (data not shown). fMLP and A23187 activation of PMN O2 generation is associated with increased [Ca2+]j; in contrast, the PMN response to PMA is not associated with changes in [Ca'"], These data suggest that the enhancing effect of influenza virus on PMN generation of O2 is "calcium-dependent." Effect of Influenza A Virus on [Ca 2+J Concentrations To determine the effect of influenza virus on [Ca2+]j, Indol-loaded PMNs were incubated with virus and changes in fluorescence monitored (Figure 6, upper panel; Table 1). After the addition of influenza virus, PMN [Ca2+]j transiently rose; in contrast, no change in [Ca2+]j was noted with PMN s incubated with noninfected allantoic fluid (Figure 6, lower panel; Table 1). The addition of tMLP (1 X 10-8 M) caused a further incr~~ in PMN [Ca2+]j, which was 21
It)
~
Q;
o 15
0 0 0
,....
8ci
to
z
(620)
~3 z w
(407)
u
(/')
~2 o
(157)
::> ..J
u,
t
of.
o
5
10
15
20
25 30 35 40 TIME (min)
45
50
55
CB
Virus
O__=.'---+----i--+--+---+--+---+--+--.---_+_~ 60 0
Figure 5. A comparison of the effect of influenza virus (virus) and non-virus-containing medium (placebo) on PMN generation of 02" by 3.0 ng PMA. Values are mean of three experiments. Standard error bars are omitted for clarity.
5
(996)
4
TABLE 1
(/')
I-
The {Ca2+j i response to jMLP following incubation with either influenza A virus or non-virus-containing culture medium, with Indo-l-loaded PMNs* Baseline [Ca2+]i
Culture Medium
Influenza Virus
fMLP (1 X 10-8 M)
108 ± 13.0t
576 ± 30t 1,214 ± 182t
Z
::> w3
u Z w u
(/')
w2
50.0 ± 4.7 53.4 ± 4.9
58.3 ± 5.8
a::
o
:3u,
t
to
Definition ofabbreviations: [Ca2+li = intracellular calcium; fMLP = formylmethionine-leucine-phenylalanine; PMN s = polymorphonuclear leukocytes. * Values (in nanomoles) are mean ± SEM; n = 4. t p < 0.05 compared to respective baseline.
CB
Placebo
o Figure 6. Upperpanel: A representative experiment showing the effect of influenza virus (virus) on [Ca-"], and the subsequent response to tMLP (1 x 10- 8 M) with Indo-l-loaded PMNs. Lower panel: A representative experiment showing the effect of a nonvirus-containing medium (placebo) on [Ca2+]jand the subsequent response to fMLP (1 x 10- 8 M) with Indo-l-loaded PMNs.
greater in medium-treated cells (Table 1). Similar experiments were conducted in the presence of ethyleneglycol-bis({3-aminoethyl ether)-N,N -tetraacetic acid (EGTA) (7 mM) to chelate extracellular Ca2+. Although there was an initial decrease in PMN fluorescence after the addition of EGTA, the influenza virus still increased PMN [Ca2+]i (Table 2). Effect of Calcium Ionophore A23187 on PMN Generation of 02" to tMLP The above observations suggested that influenza A virus "primes" the PMN for activation by the chemotactic peptide fMLP, and this effect may be caused by a nonactivating rise in [Ca"], To simulate such conditions, PMNs were incubated with 0.01, 0.1, and 1.0 #LM concentrations of A23187
[ca2+)i : 9 4
in the absence of CB. Under these conditions, calcium ionophore did not generate 02" (Figure 7). However, when fMLP (1 x 10-8 M) was then added to the incubation mixture (in the presence of CB to parallel conditions of experiments with influenza virus), PMNs that had been incubated with either 0.1 or 1.0 ItM A23187 generated more O2 than the response to fMLP alone (Figure 7). Furthermore, when
TABLE 2
The PMN {Ca2+}; response to influenza A virus or non-virus-containing culture medium and then jMLP, in the presence of 7 mM EGTA with Indo-l-loaded PMNs* Baseline
55.0 ± 6.3 54.0 ± 5.3
EGTA
Culture Medium
21.6 ± 1.7 21.7 ± 1.4
23.0 ± 2.6
Influenza Virus
64.3
± 11.7t
fMLP (1 X 10-8 M)
559 ± 66.0t 315 ± 36.8U
Definition of abbreviations: [Ca 2+] ; = intracellular calcium; fMLP = fonnyl-methionine-Ieucine-phenylalanine; EGTA = ethyleneglycol-bis-(J3-aminoethyl ether)N,N'-tetraacetic acid. * Values (in nanomoles) are mean ± SEM; n = 3. t p < 0.05 compared to baseline. p < 0.05 compared to influenza virus.
*
Busse, Vrtis, Steiner et al.: Influenza Virus Primes Neutrophils
(n= 3, mean values)
12
351
5
(+CoI.I.O~Ml
•
en
~IO
4
:: 8 Ql a.
3
o ~
A=Control (660)
Ql
~ 6
e
2
Ql
a. ~ 4
[co+ 2 Ji
en Ql
"0
=87nM
t
E 2 c
Buffer
o
t
coror Buffer (OOI.O.I.1.0~M)
Figure 7. A comparison of the effect of a preincubation with 0.01, 0.1, or 1.0 JLM calcium ionophore A23187 (Cal) or buffer on the subsequent generation of O 2 from PMNs by fMLP (1 x 10- 7 M) in the presence of 5 JLg/ml cytochalasin B. Values are the mean ± SEM; n = 5. Standard error bars are omitted for clarity.
fluorescence was monitored with Indo-l-loaded cells, a [Ca2+]j increase occurred only in PMNs that had been incubated with either 0.1 or 1.0 ILM A23187, concentrations that enhanced FMLP generation of O 2 (Figure 8).
Discussion After an in vitro incubation with live influenza virus, human PMN s generated more 0; when subsequently activated by either the chemotactic peptide fMLP or calcium ionophore A23187. With the use of a fluorescent marker to monitor intracellular concentrations of Ca2+, we found influenza virus caused a transient increase in [Ca2+1 that was not associated with O2 generation. Furthermore, in a simulated model for this virus response, incubation of PMN s with selected concentrations of A23187 that did not generate O2 resulted in increased PMN [Ca2+];; however, when A23187treated cells were subsequently activated by fMLP, O2 generation was enhanced. Collectively, these data suggest that small, nonactivating (as measured by O2 generation) concentrations of influenza virus increased [Ca2+]; to prime the PMN and lead to an increased respiratory burst when subsequently exposed to an appropriate stimulus. The effect of respiratory viruses, particularly influenza, on PMN function has been of interest to many investigators, including ourselves. When Mills and colleagues (35) incubated influenza virus with PMN s, oxygen consumption, chemiluminescence, and the generation of O2 occurred. Although Mills and colleagues (35) performed their studies in the absence of serum, we also demonstrated that influenza virus elicited a PMN chemiluminescence response that was serum-dependent, proportional to the virus-antibody titer, and associated with consumption of complement during incubation with influenza virus (34, 36). In the present study, the concentrations of influenza virus that caused the enhanced response to fMLP did not, by themselves, stimulate O2 generation (Figure 1). The difference between our current observations and those previously reported (34, 36) suggest that chemiluminescence may be a more ..sensitive
5
en
B
~4
CalCium Ionophore A23186 OOlfLM
Z
:) 3
(506)
~
Z
L&J2 U
en
(122)
I L_~-----_-:---~
CB
Col (OOlfLMl
o
:)5 ..J
IJ..
t
t
L&J
a::
c- Calcium Ionophore A23186
4
0.1 fLM (719)
3
2
I
[CO+2)=91 nM
o
200
SECONDS Figure 8. One representative experiment comparing the effect of buffer (A = control) and two concentrations of calcium ionophore A23187 (B = 0.01 JLM, and C = 0.1 JLM) on PMN [Ca2+]; and the subsequent response to fMLP (1 x 10- 8 M).
method to detect initiation of the respiratory burst and, also, that the respiratory burst to influenza virus is atypical in that H20 2 (detected by chemiluminescence) is generated but O2 is not (37). Moreover, if PMNs were incubated with large concentrations of influenza, the subsequent response of the leukocyte to an activator was depressed and paralleled previous findings noted by us and others (35-38). Thus, the resultant effect of influenza virus on PMN function is dose dependent: large concentrations of virus suppress cell activity (34, 38, 39), while smaller, nonactivating inocula prime the cell to a subsequent stimulation by selected agonists. Mechanisms for the priming of neutrophils has considerable importance to host defense and inflammation. In previ-
352
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991
ously reported studies, Koenderman and co-workers (40) exposed isolated PMNs to subactivating concentrations of platelet-activating factor (PAF) or ionomycin; when these cells were subsequently activated by tMLP, granulocyte oxygen consumption was significantly enhanced. In these experiments, the priming effect of PAF and ionomycin was associated with a transient increase in PMN [Ca'"]; Depletion of PMN Ca2+ prevented priming by either PAF or ionomycin. Our observations also imply that priming of PMNs by influenza virus is associated with an increase in [Ca"']; Concentrations of influenza virus that prime the cell increase intracellular levels of Ca 2+ without causing PMN activation, as assessed by O2 generation; in contrast, priming did not occur when influenza virus did not increase PMN [Ca2+];. Thus, like PAF (40), the priming effect of influenza virus is associated with a transient increase in PMN [Ca2+];. Because the [Ca2+]i response to influenza virus also occurred when extracellular Ca2+ was chelated by EGTA, it is assumed that the transient increase in [Ca"], arises from intracellular sources rather than an extracellular influx. However, confirmation that shifts in neutrophil calcium levels are the cause of the influenza-enhanced response need further characterization. Our studies did not determine specifically how influenza virus influenced PMN [Ca'"]; Henricks and associates (41) found neuraminidase, from either influenza virus or Vibrio cholerae, partially removed PMN membrane sialic acid and enhanced O2 generation to opsonized Staphylococcus aureus; the investigators proposed that neuraminidase unmasks membrane receptor sites to increase the efficiency of the granulocyte's metabolic burst to particle activation. Furthermore, Langer and colleagues (42) described increased calcium exchangeability when neuraminidase removed sialic acid from cultured heart cells. Because we did not measure PMN membrane sialic acid after incubation with influenza virus, the contribution of neuraminidase to the enhanced response could not be determined. If the observations by Langer and colleagues (42) relate to the PMN, it is additional support for a link between the virus effect and altered [Ca2+]; concentrations. A variety of other biologically active compounds prime human neutrophils. VanEpps and, Garcia (43) exposed human PMNs to C5 fragments and found enhanced chemiluminescence upon subsequent stimulation with dissimilar soluble and particulate activators. Because influenza virus was incubated with fresh autologous serum, it is possible that C5 fragments were generated to cause, or contribute to, PMN priming (34). However, if C5 was formed in our experiments, it was not in concentrations sufficient to generate O2• Furthermore, because we did not determine specific antibody to the influenza virus in the sera, the pathway by which the virus interacts with the neutrophil might be quite different from subject to subject. If this is the case, the end result of an enhanced neutrophil response is similar as the relative increase in O 2 generation is comparable between individual subjects. Other processes may also participate in the priming process. For example, Weisbart and associates (44) and Zimmerli and co-workers (45) found increased receptors to the
chemotactic peptide tMLP on primed PMN s. Quantitation of tMLP receptors was not done in our study, but it is unlikely that increased membrane receptors could also explain the enhanced response to ionophore. Further, PMN priming may amplify stimulus-response coupling from the receptor to the measured cellular action; Kuhns and colleagues (46) found ATP-induced enhancement of O2 generation was associated with a shortened lag time in the neutrophil response to tMLP without a change in the median effective dose for the chemotactic factor. These observations suggest that signal transduction, not altered receptor affinity, can cause enhanced oxidative activity. Parallel to our observations, Kuhns and colleagues (46) found that ATP-induced priming was associated with a rise in PMN [Ca2+]; without cell activation. Finally, Hughes and co-workers (47) and Guthrie and associates (48) suggest that either increased NADPH oxidase levels, or its activity, contributes to neutrophil priming. The effects of influenza virus on NADPH oxidase and other enzymes were not determined. Our observation that respiratory viruses enhance PMN phlogistic activity complements reports demonstrating that these microorganisms promote selected inflammatory responses. When human leukocytes are incubated with respiratory viruses, IgE-dependent basophil histamine release increases, an effect mediated, in part, by the generation of interferon (17, 18). Enhanced basophil histamine release also occurs during in vivo viral respiratory infections (19) to corroborate the potential validity of the in vitro modeling of this response. Further, if human leukocytes are incubated with respiratory viruses, basophils, but not other leukocytes, have greater chemotactic response to complement particles and lymphocyte-derived proteins (49). Finally, viral respiratory infections (11) and invitro incubations with viruses (12, 13) depress {j-adrenergic regulation of lysosomal enzyme release, a consequence that translates to promotion of PMNdriven inflammation. Although it remains to be determined precisely how, and if, toxic oxygen radicals contribute to airways injury in asthma, there is sufficient evidence that these products are injurious to the lung in other pulmonary diseases (50, 51). Our in vitro studies indicate yet another mechanism by which respiratory viruses can compound their ability to injure the airway either directly or by generation of more toxic oxygen products. Finally, although the significance of these in vitro observations to virus-provoked asthma has yet to be fully established, they suggest the following clinical possibility and relationship to the development of late asthmatic reactions (Figure 9). During viral respiratory illnesses, neutrophils become primed. Consequently, recruitment of leukocytes to the airways during allergic reactions will include primed neutrophils that have a greater inflammatory potential. When the primed PMN is activated (possibly by substances such as leukotriene B4) , the resultant bronchial inflammation is greater than nonviral respiratory illness periods and hence the probability for late asthmatic reactions increased. Because similarities have already been found between in vivo and in vitro virus effects on leukocyte function (11, 12, 18, 19), we feel that the proposed events suggested in Figure 9 are a possibility. However, these studies were conducted with
Busse, Vrtis, Steiner et al.: Influenza Virus Primes Neutrophils
The Effect of Respiratory Viruses on PMN Function
PMN~ --\.d
+ Respiratory Virus
Activation
© ~
~ca ~
c onsequence on Airways Inflammation Hyperresponsiviness Obstruction
Figure 9. A hypothetical scheme showing the possible effect of a respiratory virus on PMN function. It is postulated that the respiratory virus primes the PMN to release more 02" when activated. If activation of the primed PMN occurs in the airway, the consequence is greater inflammation, obstruction, and hyperresponsiveness.
leukocytes from normal individuals; whether similar responses will be noted with neutrophils from allergic or asthma subjects will be of considerable interest and relevance to the.issue of virus-induced reactive airways disease. Future studies, therefore, will be needed to establish this possibility and the precise mechanism by which respiratory viruses prime the neutrophil. Acknowledgments: This work was supported by Grants AI-15685, AI-23181, AI-I0404, and HL-44098 from the National Institutes of Health.
References 1. Frick, W. E., and W. W. Busse. 1988. Respiratory infections: their role in airway responsiveness and pathogenesis of asthma. Clin. Chest Med. 9:539-549. 2. McIntosh, K., E. F. Ellis, L. S. Hoffman, T. G. Lybass, J. J. Eller, and V. A. Fulginiti. 1973. The association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children. l. Pediatr. 82:578-590. 3. Minor, T. E., E. C. Dick, A. N. DeMeo, J. J. Ouellette, M. Cohen, and C. E. Reed. 1974. Viruses as precipitants of asthmatic attacks in children. lAMA 227:292-298. 4. Minor, T. E., E. C. Dick, J. W. Baker, J. J. Ouellette, M. Cohen, and C. E. Reed. 1976. Rhinovirus and influenza type A infections as precipitants of asthma. Am. Rev. Respir. Dis. 113:149-153. 5. Hudgel, D. W., C. Langston, Jr., J. C. Selner, and K. McIntosh. 1979. Viral and bacterial infections in adults with chronic asthma. Am. Rev. Respir. Dis. 120:393-397. 6. Empey, D. W., L. A. Laitinen, L. Jacobs, W. M. Gold, andJ. A. Nadel. 1976. Mechanisms of bronchial hyperreactivity in normal subjects after upper respiratory tract infection. Am. Rev. Respir. Dis. 113:131-139. 7. Saban, R., E. C. Dick, R. I. Fishleder, and C. K. Buckner. 1987. Enhancement by parainfluenza 3 infection of contractile responses to substance P and capsaicin in airway smooth muscle from the guinea pig. Am. Rev. Respir. Dis. 136:586-591. 8. Jacoby, D. B., J. Tamaoki, D. B. Borson, andJ. A. Nadel. 1988. Influenza infection causes airway hyperresponsiveness by decreasing enkephalinase. J. Appl. Physiol. 64:2653-2658. 9. Dusser, D. J., D. B. Jacoby, T. D. Djokic, I. Rubinstein, D. B. Borson, and J. A. Nadel. 1989. Virus induces airway hyperresponsiveness to tachykinins: role of neutral endopeptidase. J. Appl. Physiol. 67: 1504-1511. 10. Szentivanyi, A. 1968. The beta-adrenergic theory ofthe atopic abnormality in asthma. J. Allergy 42:203-223. 11. Busse, W. W. 1977. Decreased granulocyte response to isoproterenol in asthma during upper respiratory infections. Am. Rev. Respir. Dis. 115:783-791.
353
12. Busse, W. W., W. Cooper, D. M. Warshauer, E. C. Dick, I. H. L. Wallow, and R. Albrecht. 1979. Impairment of isoproterenol, H2 histamine, and prostaglandin E 1 response of human granulocytes after incubation in vitro with live influenza vaccines. Am. Rev. Respir. Dis. 119:561-569. 13. Busse, W. W., C. L. Anderson, E. C. Dick, and D. Warshauer. 1980. Reduced granulocyte response to isoproterenol, histamine, and prostaglandin E 1 after in vitro incubation with rhinovirus 16. Am. Rev. Respir. Dis. 122:641-646. 14. Welliver, R. C., D. T. Wong, M. Sun, E. Middleton, Jr., R. S. Vaughan, and P. L. Ogra. 1981. The development of respiratory syncytial virus-specific IgE and the release of histamine in nasopharyngeal secretions after infection. N. Engl. J. Med. 305:841-846. 15. Welliver, R. C., D. T. Wong, E. Middleton, Jr., M. Sun, N. McCarthy, and P. L. Ogra. 1982. Role of parainfluenza virus-specific IgE in pathogenesis of croup and wheezing subsequent to infection. J. Pediatr. 101:889-896. 16. Welliver, R. L., M. Sun, D. Rinaldo, and P. L. Ogra. 1986. Predictive value of respiratory syncytial virus-specific IgE responses for recurrent wheezing following bronchiolitis. J. Pediatr. 109:776-780. 17. Ida, S., J. J. Hooks, R. P. Siranganian, and A. L. Notkins. 1977. Enhancement of IgE-mediated histamine release from human basophils by viruses. Role of interferon. J. Exp. Med. 145:892-896. 18. Busse, W. W., C. A. Swenson, E. C. Borden, M. W. Treuhaft, and E. C. Dick. 1983. Effect of influenza A virus on leukocyte histamine release. J. Allergy Clin. Immunol. 71:382-388. . 19. Lemanske, R. F., Jr., E. C. Dick, C. A. Swenson, R. F. Vrtis, and W. W. Busse. 1989. Rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions. J. Clin. Invest. 83: 1-10. 20. deMonchy, J. G. R., H. F. Kauffman, P. Venge etal. 1985. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions. Am. Rev. Respir. Dis. 131:373-376. 21. Metzger, W. J., H. B. Richerson, K. Worden, M. Monick, and G. W. Hunninghake. 1986. Bronchoalveolar lavage of allergic asthmatic patients following allergen provocation. Chest 89:477-483. 22. Carroll, M. P., S. R. Durham, G. Walsh, andA. B. Kay. 1985. Activation of neutrophils and monocytes after allergen- and histamine-induced bronchospasm. J. Allergy Clin. Immunol. 75:290-296. 23. Murphy, K. R., M. C. Wilson, C. G. Irvin et al. 1986. The requirement for polymorphonuclear leukocytes in the late asthmatic response and heightened airways reactivity in an animal model. Am. Rev. Respir. Dis. 134:62-68. 24. Fabbri, L. M., P. Boschetto, E. Zocca et al. 1987. Bronchoalveolar neutrophilia during late asthmatic reactions induced by toluene diisocyanate. Am. Rev. Respir. Dis. 136:36-42. 25. Metzger, W. J., G. W. Hunninghake, and H. B. Richerson. 1985. Late asthmatic responses: injury inquiry into mechanisms and significance. Clin. Rev. Allergy 3:145-165. 26. O'Byrne, P. M., J. Dolovich, and F. E. Hargreave. 1987. Late asthmatic responses. Am. Rev. Respir. Dis. 136:740-751. 27. Lemanske, R. R., Jr., and M. A. Kaliner. 1988. Late-phase allergic reactions. In Allergy: Principles and Practice. E. Middleton, Jr., C. E. Reed, E. F. Ellis, N. F. Adkinson, Jr., and J. W. Yanginger, editors. C. V. Mosby, St. Louis, MO. 224-246. 28. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21(Suppl. 97):77-89. 29. Sedgwick, J. B., R. F. Vrtis~/M. F. Gourley, and W. W. Busse. 1988. Stimulus-dependent differences in superoxide anion generation by normal eosinophils and neutrophils. J. Allergy Clin. Immunol. 81:876-883. 30. Pick, E., and D. Mizel. 1981. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. J. Immunol. Methods 46:211-226. 31. Lazzari, K. G., P. S. Proto, and E. R. Simons. 1986. Simultaneous measurement of stimulus-induced changes in cytoplasmic Ca2+ and in membrane potential of human neutrophils. J. Bioi. Chem. 261:9710-9713. 32. Nasmith, P. E., and S. Grinstein. 1987. Are Ca 2+ channels in neutrophils activated by a rise in cytosolic Ca2+? FEBS Lett. 221:95-100. 33. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. A new generation of Ca 2+ indicators with greatly improved fluorescence properties. J. BioI. Chem. 360:3440-3450. 34. Shult, P. A., E. C. Dick, K. A. Joiner, and W. W. Busse. 1985. Role of serum in stimulation of polymorphonuclear leukocyte, luminol-dependent chemiluminescence by influenza A. Am. Rev. Respir. Dis. 131:267-272. 35. Mills, E. L., Y. Debets-Ossenkopp, H. A. Verbrugh, and J. Verhoef. 1981. Initiation of the respiratory burst of human neutrophils by influenza virus. Infect. Immun. 32:1200-1205. 36. Busse, W. W., andJ. M. Sosman. 1981. Altered luminol-dependent granulocyte chemiluminescence during an in vitro incubation with influenza vaccine. Am. Rev. Respir. Dis. 123:654-658. 37. Hartshorn, K. C., M. Collamer, M. R. White, J. H. Schwartz, and A. I. Tauber. 1990. Characterization of influenza A virus activation of the human neutrophil. Blood 75:218-226. 38. Faden, H., P. Sutyla, and P. Ogra. 1979. Effect of viruses on luminol-
354
39.
40. 41.
42. 43. 44.
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991
dependent chemiluminescence of human neutrophils. Infect. Immun. 24:673-678. Cassidy, L. F., D. S. Lyles, and J. S. Abramson. 1989. Depression of polymorphonuclear leukocyte functions by purified influenza virus hemagglutinin and sialic acid-binding lectins. J. Immunol. 142:44014406. Koenderman, L., Yazdanbakhsh, D. Roos, and A. J. Verhoeven. 1989. Dual mechanisms in priming of the chemoattractant-induced respiratory burst in human granulocytes. J. Immunol. 142:623-628. Henricks, P. A. J., M. E. van Erne-van der Tol, and J. Verhoef. 1982. Partial removal of sialic acid enhances phagocytosis and the generation of superoxide and chemiluminescence by polymorphonuclear leukocytes. J. Immunol. 129:745-750. Langer, G. A., J. S. Frank, L. M. Nudd, and K. Seraydavin. 1976. Sialic acid: effect of removal on calcium exchangeability of cultured heart cells. Science 193:1013-1015. VanEpps, D. E., andM. L. Garcia. 1981. Enhancement of'neutrophil function as a result of prior exposure to chemotactic factor. J. Clin. Invest. 66:167-175. Weisbart, R. H., D. W. Golde, and J. C. Basson. 1986. Biosynthetic human GM-CSF modulates the number and affinity of neutrophil f-met-leuphe receptors. J. Immunol. 137:3584-3587.
45. Zimmerli, W., B. E. Seligmann, and 1. I. Gallin. 1987. Neutrophils are hyper-polarized after exudation and show an increased depolarization response to formyl-peptide but not to phorbol myristate acetate. Eur. J. Clin. Invest. 17:435-441. 46. Kuhns, D. B., D. G. Wright, J. Nath, S. S. Kaplan, and R. E. Basford. 1988. ATP induces transient elevations of [Ca2+]i in human neutrophils and primes those cells for enhanced 02" generation. Lab. Invest. 58:448-453. 47. Hughes, V., J. M. Humphreys, and S. W. Edwards. 1987. Protein synthesis is activated in primed neutrophils: a possible role in inflammation. Biosci. Rep. 7:881-890. 48. Guthrie, L. A., L. C. McPhail, P. M. Henson, and R. B. Johnston, Jr. 1984. Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide. J. Exp. Med. 160:1656-1671. 49. Lett-Brown, M. A., M. Aelvolet, J. J. Hooks, J. A. Georgiades, D. O. Thueson, and J. A. Grant. 1981. Enhancement of basophil chemotaxis in vitro by virus-induced interferon. J. Clin. Invest. 67:547-552. 50. Fantone, J. C., D. E. Feltner, J. K. Brieland, and P. A. Ward. 1987. Phagocyte cell-derived inflammatory mediators and lung disease. Chest 91:428-435. 51. Heffner, J. E., and J. E. Repine. 1989. Pulmonary strategies of antioxidant defense. Am. Rev. Respir. Dis. 140:531-554.
