pulmonary surfactant.1 Surfactant deficiency results in alveolar collapse at ... These phospholipid-protein complexes could conceiv- ably be antigenic when ...
Surfactant-A nti-Surfactant Immune Complexes With
Respiratory Distress
in
Infants
Syndrome
DAVID S. STRAYER, MD, PhD, T. ALLEN MERRITT, MD, JAMESON LWEBUGA-MUKASA, MD, and MIKKO HALLMAN, MD
The authors sought to determine whether treatment of respiratory distress syndrome (RDS) with human surfactant resulted in the formation of detectable circulating immune complexes. Preterm infants with severe RDS were divided into two groups: one group received human surfactant by intratracheal instillation and the other group did not. Both groups received ventilatory management involving intermittent mandatory ventilation. Plasma samples were drawn from these babies prior to treatment and at intervals thereafter. The authors developed an ELISA assay specific for surfactant-anti-surfactant immune complexes and analyzed the plasma samples for such immune complexes. Complement levels were also measured. They found that with time plasma from RDS infants in both groups showed evidence of sur-
RESPIRATORY distress syndrome (RDS) occurs in preterm infants with immature lungs who are deficient in pulmonary surfactant.1 Surfactant deficiency results in alveolar collapse at end-expiration and progressive ventilatory failure. Heterologous surfactant administration,2'3 artificial surfactant,4 and surfactant derived from human amniotic fluid5 have been shown to improve arterial oxygen tension and reduce the need for conventional ventilatory assistance in neonatal RDS. These phospholipid-protein complexes could conceivably be antigenic when administered intratracheally,
From the Departments of Pathology and Medicine, Yale University School of Medicine, New Haven, Connecticut; Department of Pediatrics, University of California, San Diego, La Jolla, California; and the Department of Pediatrics, Children's Hospital, University of Helsinki, Helsinki, Finland
factant-anti-surfactant immune complex formation. The concentrations of immune complexes generally peaked within the first week of life and then appeared to diminish over 1-4 weeks after birth in RDS infants. There was no evidence at any time in either group of immunecomplex-mediated injury or of decreased serum complement levels. It is concluded that circulating immune complexes between surfactant and antibodies to surfactant are probably found in most neonates with respiratory distress syndrome, that they do not produce pulmonary damage detectable by clinical and serologic means, and that treatment of neonatal RDS with human surfactant similarly does not produce lung injury as determined with these techniques. (Am J Pathol 1986, 122:353-362)
Supported in part by the U.S. Public Health Service CA16136 (Dr. Strayer), HD10622, HD16292 and the March of Dimes (Dr. Merritt), and the Finnish Academy of Sciences (Dr. Hallman). Dr. Strayer was the recipient of American Cancer Society Junior Faculty Clinical Fellowship JF CF637 while part of this work was being performed. These data were presented in preliminary form at the annual meeting of the Society for Pediatric Research, San Francisco, California, in May 1984. Accepted for publication September 25, 1985. Address reprint requests to David S. Strayer, MD, PhD, Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510.
353
354
STRAYER ET AL
AJP * February 1986
Table 1 -Summaries of Clinical Histories
Case
Gestational
number
age (weeks) 30 28 42
2 3
30 30 27 29 31
4 5
6 7
8 9 10
Sex M
M F
F M F F
M F
M
27
11
12 13 14 15 16 17
F M
32 31 30
M F
M M
34
F
Birth weight (gm)
Presence of RDS
1260 920 3500
+ +
1480 1000 1160 1400 1240 1460 1070 850 900 1660 1080 1140 860 1400
+ + + + + + + + + + + +
SRF treatment
Other medical problems PDA, IVH PDA, IVH Meconium aspiration, pulmonary hypertension PDA, PIE PDA, IVH PDA, IVH PDA IVH IVH PDA PDA, IVH PDA PIE, IVH PDA Pneumonia, chorioamnionitis PDA, NEC
Brief summaries of clinical histories of patients included in this study are shown above. We included only patients for whom we had adequate plasma samples for testing for immune complexes over a time course of 7 days or more. PDA, patient ductus arteriosus; PIE, pulmonary interstitial emphysema; IVH, intraventricular hemorrhage; NEC, necrotizing enterocolitis.
thus raising concerns about the potential for sensitization or immunologic injury resulting from immunecomplex formation in infants so treated.6 We set out to determine whether circulating surfactant-antisurfactant immune complexes were detectable in infants treated with human surfactant (SRF) and whether they caused clinically detectable injury.
Materials and Methods Surfactant
Surfactant was isolated from human term amniotic fluid obtained at cesarean section by density gradient ultracentrifugation as detailed elsewhere.5 Human SRF contains 4-5 %o protein. These preparations contain an apoprotein component and attached phospholipid moieties. The SRF apoprotein is approximately 34,000 daltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.7*
Treatment
The clinical and radiographic features of severe RDS, details of the experimental protocol used, and the technique of human surfactant administration in very low birthweight infants, have been detailed by Hallman et Human surfactant is most likely a heterogeneous group of molecules, of differing molecular weights and degrees of *
glycosylation. Understanding this, tant" for the sake of simplicity.
we use
the term "surfac-
al.5 Briefly, infants received a 60-mg/kg body weight SRF by intratracheal instillation by 10 hours after birth when severe RDS (FiO2 > 0.6) was evident and mechanical ventilation failed to increase the PaO2 > 60. A comparison group of infants with severe RDS was allocated with random assignment to conventional ventilatory management alone. Those receiving surfactant and those receiving conventional ventilation formed the study groups. We also studied sera from preterm infants without lung disease to determine the effect of lung disease on immune complex formation. After informed consent for allocation into study groups, plasma samples were obtained from all treatment groups in accordance with approved procedures. Serum complement analyses (CH50, C3, C3a and Clq binding) were performed. Urine was examined daily for blood and protein by routine techniques. Each infant was carefully evaluated for the presence of skin lesions or rashes, and clinically indicated chest radiographs were evaluated as previously described. A summary of case histories is provided (Table 1). Two representative case histories are included in the Appendix. Complete case histories are available on request from the authors. Cases 1, 3-6, and 15 received no SRF. Case 17 was a premature infant with no RDS. All other infants were premature infants with RDS who received SRF.
Antibody to Surfactant Antibody to human SRF preparations was prepared in rabbits according to the following protocol. Thirty micrograms of surfactant was emulsified in Freund's
IMMUNE COMPLEXES IN RDS
Vol. 122 * No. 2
complete adjuvant (Difco) and injected into 3-kg female New Zealand white rabbits (Red Beau Farms, Jamul, Calif) at four sites subcutaneously in the back. The first boost with surfactant was administered similarly, the dose being 10 Mg, in incomplete Freund's adjuvant (Difco). Additional injections were performed with 5-7.5 ,ug SRF given intravenously in saline. Rabbits were bled 7-10 days after injection. All rabbit sera were kept separate according to each rabbit and the date. Rabbits were bled before the initial injection of SRF. All sera were titered for anti-SRF activity by enzymelinked immunosorbant assay (ELISA, see below). Murine antibodies to surfactant were raised in a similar fashion in DBA/2J mice (Jackson Laboratory) with a primary dose of 5 jig surfactant in adjuvant followed by 1 ,ug intravenous boosts in saline. Mice were bled from the retroorbital sinus, and their sera were pooled. These sera were assayed for anti-SRF activity following serial double dilution in phosphate-buffered saline. Surfactant bound to ELISA plates (Dynatech) at 200 ng/well was incubated with the antisera. Horseradish peroxidase-labeled goat anti-rabbit Ig or goat antimouse Ig (Miles Pharmaceuticals) was added, followed by substrate (see below). Goat antibodies to human IgG, gamma chain specific, were the kind gift of Dr. James McNamara in Dr. John Dwyer's laboratory (Department of Medicine, Yale University School of Medicine). This antibody preparation was not conjugated to enzyme. Assay for Surfactant-Anti-Surfactant Immune Complexes We developed an assay specific for SRF-anti-SRF immune complexes, following standard ELISA techniques.8 This assay is illustrated in Figure 1. Briefly, round-bottom ELISA plates (Dynatech) were coated with rabbit anti-human surfactant antiserum (10 jig protein/well) and washed. One high titer sample of rabbit antiserum was chosen and used exclusively. The human plasma samples in question were diluted and added to the wells, which were incubated and then washed. Such a procedure would bind any circulating surfactant in the neonatal plasma. Heavy-chain-specific, rabbit antihuman IgG that had been conjugated to horseradish peroxidase (HPO-anti-human IgG, Miles Pharmaceuticals) was added. After washing, bound enzymatic activity was measured with the use of ortho-phenylendiamine (OPD, Sigma Chemical Co.) in an ELISA reader (Dynatech) at 490 nm. Some determinations were performed using alkaline phosphatase-conjugated secondary antibodies (Miles Pharmaceuticals). The substrate used for alkaline phosphatose was p-nitrophenyl amine (Sigma Chemical Co.); absorbance was read at 405 nm.
355
wash SRF
hu ant
incubate
HPO -conjugated rabbi t anti -human Ig HPO_ HPO conjugated rabbit anti -human Ig
wash immune
complexes bind
rabbit anti- SRF
incubate
H PO;
I
add substrate ( OPD)
H202 IH PO
wash
rabbit anti -human Ig binds the human Ig complexed to SRF
HOj>
peroxidose bound to the immune complex converts the substrate to a colored reaction product incubote, then stop reaction with 4N H2SO4
A-HPOI colored reaction extent of binding measured as A490
Figure 1 -The procedure for performing the ELISA for SRF-anti-SRF immune complexes.
Specificity of the Antibody Preparations In a series of experiments, rabbit anti-SRF antibody was thrice absorbed with Sepharose 4B (Pharmacia) conjugated to normal human Ig (the kind gift of Dr. Harry Bluestein, Department of Medicine, University of California, San Diego, Calif). The same rabbit antiserum was absorbed in parallel with unconjugated Sepharose 4B as a volume control. ELISA plates were coated with these absorbed preparations as usual and tested in parallel versus a panel of serially double-diluted neonatal plasma samples for binding activity for
356
STRAYER ET AL
SURFACTANT
AJP
SERUM
*
February 1986
serum. Rabbit anti-SRF, or normal rabbit serum, was added to these sections. This was followed by FITCgoat anti-rabbit Ig (Cappel Laboratories). Reactivity was measured on a Leitz Fluorescence microscope using Pleom optics. In control sections, normal rabbit serum was substituted for rabbit anti-SRF.
67+ Complement The third component of complement and C3a were measured in sera taken at 14 days of age with the use of radial immunodiffusion (Milroy Laboratories, Springfield, Va), and total hemolytic complement ; (CH50) was also measured by standard techniques for determination of the degree of activation of the classical complement pathway. The binding of Clq was measured at a reference laboratory.
33+29+
14+
Analysis
A B
A B
Figure 2-Western blots of human surfactant (/eft) and human serum (right). A-Staining with the absorbed anti-SRF antiserum followed by HPO-anti-rabbit IgG. B-Staining with 0.1% amino black.
SRF-anti-SRF immune complexes. Raw absorbance values for each plasma sample were compared for both groups with the use of a paired-observation t test. We found that prior absorption of the rabbit antiserum with human IgG did not alter the binding observed in neonatal plasma (0.40 > P > 0.30). Thus, the rabbit anti-SRF antiserum we used did not bind normal human Ig enough to produce a false-positive result. Nonetheless, before being used in immune complex assays, rabbit antisera were absorbed with human plasma proteins conjugated to Sepharose 4B. Western Blots Western blots were performed according to standard protocol.9 Human SRF, from the autopsy lung of an infant with no pulmonary disease, and normal adult human serum were separated by SDS-PAGE and transferred to nitrocellulose filters. These filters were treated with a blocking agent, then exposed to the rabbit antiSRF antiserum, followed by HPO-goat anti-rabbit IgG and OPD. Immunofluorescence Assay Cryostat-cut frozen sections of normal human lung were used. These were first incubated with normal goat
Differences in complement activities among groups compared with the use of a nonpaired Student t
were
test.
Results Specificity of Rabbit Anti-Surfactant Antiserum The specificity of the rabbit antiserum to human SRF was examined with two procedures. The first was Western blot (Figure 2). The major reduced apoprotein portion of SRF has a molecular weight of 34 kd.' The proteins of human SRF, separated by SDS-PAGE, revealed two major components under reducing conditions, 68 and 33 kd, and two smaller components of 28 and 14 kd. Only the 68 kd protein did not have immunoreactivity against the absorbed rabbit anti-human SRF antiserum. The same antiserum had no detectable immunoreactivity against normal human serum. Immunofluorescence was also used to determine the localization within the lung of reactivity in the antiSRF preparations (Figure 3). Frozen sections of lung stained with the antiserum show granular staining of scattered cells along alveolar walls. Type I alveolar cells, interstitial components, and blood do not stain with these techniques. Thus, the pattern of immunofluorescence reactivity seen here confirms that the rabbit antiSRF antiserum recognizes only granular structures present in the cytoplasm of Type II pneumocytes. Similarly, we observed comparable staining of A549 cells, a human tumor line composed of Type II pneumocytes. Interpretation of the immune complex assay requires credible establishment of the lack of reactivity of our antisera with normal human Ig. To investigate further
IMMUNE COMPLEXES IN RDS
Vol. 122 * No. 2
whether our rabbit anti-SRF antibody showed antibody activity against human Ig, we coated ELISA plates with anti-SRF antiserum (previously absorbed three times with human plasma), normal rabbit serum (unabsorbed), and goat anti-human IgG. The former two sets of plates were coated with their respective rabbit sera at ten times the usual concentration (10o in coating mixture versus 0.10/o) used in performing the immune complex determinations. This was done to permit us to detect even minor reactivity to human Ig, undetected by immunofluorescence or Western blot and unlikely to be detected by the ELISA for immune complexes. Specific binding of human Ig to these three different coated plates was determined (Figure 4). The results indicated that the rabbit anti-SRF preparation used for the SRF-anti-SRF immune complex ELISA does not demonstrably bind human Ig.
10
357
r
0.9 -
0.8
X.. I
0.7 F
%,-
-
x-_
_
__X
I
I
0.6
A45 0.5
'x I I
I I~~~~
nt-P raT-n
0.4: 0 NRS
0.3
x Ganti -HIgG
0.2
).1I\
0
1:10
1:20
1:40 1:80 1:160 1:320 1:640 1:1280 DILUTION of HUMAN Ig
Figure 4-Specific binding of human Ig to ELISA plates bearing goat antihuman IgG, normal rabbit serum, and rabbit anti-SRF. To detect even slight Hig binding activity undetectable with the use of our immune complex assay, concentrations of rabbit anti-SRF and normal rabbit serum used to coat these plates were ten times those used for determinations of circulating SRF-anti-SRF immune complexes. The normal rabbit serum is not previously absorbed versus Hlg. Rabbit anti-SRF is previously absorbed versus HIg. The initial concentration of human lg is 6.12 mg/ml.
ELISA Assay We developed the ELISA technique employed here in order to measure specific immune complexes formed between SRF and human anti-SRF antibody. Other immune complexes or noncomplexed Ig should not be detected. The specificity of the assay was ascertained with the use of artificially generated murine immune complexes. Serially diluted murine anti-SRF antiserum was mixed with 100 ng human SRF. Normal mouse serum was used as the negative control. These preparations were assayed on ELISA plates coated with rabbit antiSRF anti-serum. The results (Figure 5) indicated that our assay is capable of detecting SRF-anti-SRF immune complexes. This assay can distinguish these from "nonspecific" binding of normal serum at dilutions as high as 1L2.7 Immune Complexes in Infants With RDS Figure 3a-Frozen section of normal human lung stained with rabbit-antiSRF and FITC-goat anti-rabbit 1g. Only Type II pneumocytes stain. Interstitium, Type pneumocytes, and basement membranes do not react with the antiserum. The staining pattern is coarsely granular in Type II pneub-same section treated identically except that we mocyte cytoplasm. used normal rabbit serum instead of rabbit anti-SRF.
In infants with RDS, both those treated with SRF and those not treated with SRF, plasma samples were evaluated at weekly intervals. Blood from two premature infants without RDS was also sampled similarly.
358
STRAYER ET AL
AJP * February 1986
2.0 1.8 0.
1.6
1.4 9,~ ~
1.2 0
4a
~
~
~
9,~~~~~" a
\b
1.0
9,. 9,
~
~
--o
'o
.
Serum Complement Activity
.8
.6 .4 .2
0
for more than 1 week or that there were inadequate sample volumes for evaluation. Infants in both the SRFtreated group and the control group showed evidence of SRF-anti-SRF immune complexes. One child without RDS but with chorioamnionitis (Case 15) showed these immune complexes. One healthy premature infant (Case 17) showed no evidence of circulating SRF-anti-SRF immune complexes. Detectable immune complexes were generally at their highest levels within 2 weeks of birth, and declined thereafter.
IL
I
il
2
3
4 log2
5
6
I
I
7
8
9
CH50 levels in SRF-treated infants were 90 ± 15 U/ml versus 105 ± 22 U/ml in infants receiving conventional therapy, whereas C3 levels in the SRF-treated infants were 49 ± 14 mg/dl versus 55 ± 17 mg/dl in the control infants. C3a levels were 21 ± 14 ng/dl and 14 ± 8 mg/dl, respectively. Clq binding was < 10OW in all infants and considered normal. None of these comparisons indicates a statistically significant difference between SRF-treated and control patients.
DILUTION
Figure 5-Sensitivity of immune complex detection with the ELISA assay. Plates were coated with rabbit anti-SRF antibody. Aliquots of pooled mouse anti-SRF antisera were serially double-diluted and added to 100 ng human SRF. These preformed immune complexes were added to ELISA plates. We used HPO-rabbit anti-mouse IgG as a secondary antibody, followed by the substrate, OPD. Immune complex binding was measured as 0) and A490 for the preformed complexes with mouse antiserum (O normal mouse serum * - -- 0). - --
Their plasma samples were tested for SRF-anti-SRF immune complexes. Though some individual variation was observed, we detected a surprisingly consistent pattern of SRF-anti-SRF complexes. That is, infants in both SRF treatment and control groups tended to develop detectable circulating SRF-anti-SRF immune complexes. These immune complexes are recorded as the ratios of absorbance at the time in question: absorbance of the pretreatment blood sample taken immediately postnatally. A490 is used for HPO-conjugated antibody; A405 is used for alkaline phosphatase-conjugated antibody. Relative absorbance is reported, rather than absolute absorbance, both to facilitate comparison of assays performed using peroxidase and akaline phosphatase-conjugated antibodies, and to accommodate variations in background observed with the use of these techniques. Detection of immune complexes is illustrated in Figure 6. The results from all infants followed for more than 1 week are shown. Differences in numbers of infants between treatment and control groups reflect the fact that in not all infants were plasma samples taken
Discussion
Lung SRF is synthesized in Type II alveolar cells and secreted into alveoli or terminal air saccules. Surfactant turnover involves recycling of the alveolar complex between alveolar lining and Type II cells, breakdown of this lipid-protein complex, and resynthesis.II Although a fraction of SRF may be transported up the tracheobronchial tree and either swallowed or expectorated, or engulfed by alveolar macrophages, it does not appear to contact the internal immune system of a normal infant. Therefore, SRF may be similar to other excretory products, such as keratins or various gastrointestinal mucosal antigens, that are known to be im-
munogenic."2,3 The immature respiratory system of the neonate, particularly the preterm infant with RDS, may represent a pathologic state in which even homologous surfactant given intratracheally could "leak" into the circulation and thus elicit an antibody response. The result, we hypothesized, could be circulating immune complexes of adequate size to produce immune-complexmediated lung, renal, or other injury. In our initial studies of SRF treatment in infants with RDS, we sought to measure circulating specific SRF-anti-SRF immune complexes over the first weeks of life, after either SRF treatment or conventional therapy. Although absolute absorbance varied greatly from one infant to the next, a relatively consistent time course of detectable immune complexes was noted. An initial climb in those complexes following the development
15
1 .3H
0
0)
aw
0)
m
o(1~ ~ ~03) (
A1
_0-
z
,
oo-
o 1.2-
i0 1,01
0 m
0f)
) -
0
\ 0Ow/N(12)
1.0
Jr
0.8.
1
naO
20
10
30
40
50
70
60
Days After Birth 0.9
4
0
8
12
16 20 24 28
DAYS 1.2
12 r z
< m 0 m
II _
