Session Information
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: Previous studies suggested that unknown plasma factors increase oxidative burst in Behçet’s disease (BD), but little is known about neutrophil extracellular traps (NET) formation. Herein, we investigated in patients with active (aBD) and inactive BD (iBD): soluble CD40L (sCD40L) plasma levels; the effect of CD40 pathway activation on NET release and oxidative burst; and the expression of CD40L and its receptors.
Methods: Neutrophils and PBMC were obtained from patients with aBD (n=30), iBD (n=31), and healthy controls (HC; n=30). For each group, a pool of plasma treated or not with recombinant CD40 (rhCD40) for sCD40L blockade was created. A pool of plasma from four CD40L-deficient patients was also obtained. sCD40L plasma concentration was measured by Luminex. NET formation was quantified on neutrophils plated onto circular coverslips, followed by labeling with Hoechst 123 and antibodies to histones and neutrophil elastase. H2O2/O2–production was determined by luminol/lucigenin luminescence intensity. Cells were treated with PMA, sCD40L or the pool of plasma. Flow cytometry analysis evaluated the expression of 1) CD40, Mac-1, α5β1-integrin and α2bβ3-integrin on neutrophils and monocytes; 2) CD40L on T cells; 3) CD40L on platelets.
Results:
sCD40L plasma levels were significantly higher in iBD (median/range=17,234/2,346-19,279) and aBD (18,288.50/412.58-19,883) compared to HC (47.45/33.68-26,743; p<0.001). NET formation was constitutively increased in BD compared to HC. Stimulation of in vitro NET formation by aBD plasma or sCD40L was inhibited by CD40L blockade (Table 1). Neutrophil and PBMC production of O2–/H2O2 was higher after stimuli with sCD40L, aBD or iBD plasma than with HC plasma and decreased after CD40L blockade (Table 2). Mac-1 expression was constitutively increased in iBD (4,615/208-8,594) and aBD (2,018/891-6,533) neutrophils compared to HC (55.35/17.5-70.5; p<0.01). CD40, CD40L, α5β1- and α2bβ3-integrin expression were similar in the three groups.
Conclusion: Plasma from aBD patients exerts a stimulus on NET formation and oxidative burst, probably mediated by Mac-1. Increased concentration of sCD40L might be associated with neutrophil hyperactivity in BD.
Table 1 – Neutrophil extracellular traps (NETs) formation
|
|
NET formation (µm2) |
||
|
HC |
iBD |
aBD |
|
|
No stimulusa |
20.00±4.08*¨ |
146.50±15.58¨«» |
207.75±51.99¨« |
|
PMA |
594.83±102.21¶ |
512.50±37.99¶ |
514.00±51.69¶ |
|
sCD40L |
836.22±206.05¶ |
1443.42±92.10¶ |
1442.00±494.48¶ |
|
Plasma from HCa |
62.33±11.91¨ |
161.83±31.80¨«» |
210.50±32.30¨« |
|
Plasma from HC + rhCD40 |
65.50±40.02 |
60.78±38.27†… |
70.617±30.56…† |
|
Plasma from iBDa |
66.75±25.86¨ |
197±34.03*¨« |
225±58.41¨« |
|
Plasma from iBD + rhCD40 |
62.98±33.41 |
87.24±29.99…† |
80.43±33.65…† |
|
Plasma from aBDa |
123.25±46.56¶ |
351±36.25¶» |
304.75±20.12¶ |
|
Plasma from aBD + rhCD40 |
71.12±30.71†… |
83.22±17.77†… |
104.50±19.90…«† |
Table 2 – Production of O2– and H2O2
|
Source of neutrophils |
No stimulusa (RLU) |
sCD40L (RLU) |
Plasma from HIgMa (RLU) |
Plasma from HCa (RLU) |
Plasma from iBDa (RLU) |
Plasma from aBDa (RLU) |
|||
|
rhCD40- |
rhCD40+ |
rhCD40- |
rhCD40+ |
rhCD40- |
rhCD40+ |
||||
|
O2– production |
|||||||||
|
Healthy control |
1,561 100 to 20,428¶ |
46,922 5,862 to 296,460¶ |
3,528 1,298 to 7,564 |
20,150 4,156 to 79,584 |
14,189 2,460 to 55,318 |
54,848 10,584 to 117,380* |
23,212 8,109 to 124,484†… |
56,996 6,880 to 232,802* |
26,142 9,202 to 112,554†… |
|
iBD |
856 44 to 41,492¶ |
68,600 10,472 to 615,780¶ |
2,903 1,003 to 7,940 |
21,954 2,472 to 83,160 |
15,386 4,058 to 63,372 |
60,943 5,024 to 273,100* |
27,098 10,034 to 165,606†… |
57,408 7,956 to 233,104* |
31,230 4,943 to 166,448†… |
|
aBD |
3,477 544 to 15,702¶ |
338,040 97,270 to 379,520*¶ |
7,093 1,439 to 8,292 |
33,648 5,836 to 93,952 |
10,256… 3,352 to 55,252† |
69,549 12,580 to 165,444* |
28,675 18,318 to 119,578†… |
61,392 12,532 to 186,668* |
27,633 13,633 to 141,624†… |
|
H2O2 production |
|||||||||
|
Healthy control |
1,607 344 to 6,216¶ |
244,833 48,636 to 358,276¶ |
3,298 1,135 to 8,082 |
39,888 5,484 to 137,424 |
34,391 3,690 to 290,354…† |
76,170 10,054 to 290,000*¨ |
20,895 5,683 to 93,590…† |
190,310 11,468 to 511,472* |
55,608 6,444 to 410,401†… |
|
iBD |
944 368 to 14,632¶ |
318,768 58,234 to 489,016¶ |
2,767 1,154 to 5,861 |
33,764 5,148 to 210,644 |
33,896 2,610 to 340,022†… |
87,432 8,020 to 341,972* |
16,168 10,546 to 245,998…† |
107,687 8,136 to 399,116* |
93,528 16,494 to 441,561†… |
|
aBD |
2,684 620 to 8,789¶ |
452,846 155,338 to 1,270,000*¶ |
8,082 2,431 to 34,956 |
49,792 6,012 to 166,704 |
38,038 2,936 to 95,320…† |
106,832 34,708 to 465,252*¨ |
47,282 5,038 to 148,932…† |
365,877 33,556 to 1,779,106* |
80,075 10,453 to 403,571†… |
RLU: relative light units; a Figures correspond to median and range or mean±SD; * p<0.05 comparing to “stimulus with plasma from HC”; ¨ p<0.05 comparing to “stimulus with aBD plasma”; ¶ p<0.05 comparing to any form of stimulus; † p<0.05 comparing to the analogous pool of plasma without rhCD40.
To cite this abstract in AMA style:
Perazzio SF, Pereira PVS, Souza AWS, Condino-Neto A, Andrade LEC. CD40/CD40L Pathway Is Associated with Increased Oxidative Burst and Neutrophil Extracellular Traps Release in Behcet´s Disease [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/cd40cd40l-pathway-is-associated-with-increased-oxidative-burst-and-neutrophil-extracellular-traps-release-in-behcets-disease/. Accessed .« Back to 2015 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/cd40cd40l-pathway-is-associated-with-increased-oxidative-burst-and-neutrophil-extracellular-traps-release-in-behcets-disease/