SDC: Material and Methods
Binding specifity of infliximab to rat TNFa:
Preliminary experiments tested infliximab as a human antibody in rats and showed high efficacy in a rat model of ITX 11. In order to confirm these data, we investigated specific binding to rat TNFa by fluorescence activated cell sorting analysis (FACS).
Infliximab (Essex, Munich, Germany) and a mouse-anti-human IgG1 mAb (BD, Heidelberg, Germany) were biotinylated with No-Weigh-Sulfo-NHS-LC-Biotin (Biotinylation kit, Pierce, Rockford, IL, USA). The mouse-anti-human IgG1 mAb served as isotype control. The binding of infliximab to TNFa, produced by human peripheral blood mononuclear cells (PBMC), served as the positive control. Infliximab and streptavidin (BD, Heidelberg, Germany) alone constituted the negative controls.
Spleens from LEW rats were harvested and single cell suspensions were prepared.
Human PBMC were isolated from peripheral blood and resuspended by a standard procedure.
Both cell suspensions stimulated with lipopolysaccharide (LPS: 10µg/ml; Sigma, Seelze, Germany) for 5h at 37°C. Unstimulated cell suspensions served as controls. Afterwards, stimulated and unstimulated cell suspensions were incubated with either 1µl of biotinylated infliximab or isotype control for 30min at 4°C. After incubation with fluorescent streptavidin-phycoerythrin conjugate (BD, Heidelberg, Germany) for 15min (4°C) suspensions were fixed with paraformaldehyde solution. Finally, the level of membrane bound infliximab in the samples was measured with a FACScalibur (Becton Dickinson, BD Heidelberg, Germany) and data analyzed using BD CellquestTM Pro Software.
Assessment of binding specificity revealed that infliximab specifically binds membrane bound TNFa of rat MNC at a similar level as human MNC (Figure 6, SDC). Binding levels were 99.66% in human and 98.76% in rat MNC, respectively (p=n.s.). Analysis of the negative controls (human and rat MNC without any additions) did not show any significant antibody binding. The low binding level of the isotype control (Isotype control (IgG1kappa)+Streptavidin: human MNC 0.81% vs. rat MNC 2.37% p=n.s.) revealed a
significant difference compared with the high binding level of infliximab in human and rat MNC, indicating that no unspecific interactions were present. Further controls with infliximab alone and streptavidin alone displayed no significant binding and no difference between the groups.
SDC, Legends
SDC, Table 1
The TX control group displayed significantly increased infiltration with inflammatory CD8+, CD4+, and CD25+ T-cells and ED1+ monocytes/macrophages in the distal portion compared with the proximal segment of the graft at all time points following reperfusion.
This difference persisted in most cases, even after TNFa blockade.
SDC, Figure 1
Immunohistolgical staining showing the expression of CD4+ and CD8+ in the proximal and distal parts of the intestinal grafts of all groups.
TNFa inhibition with infliximab, etanercept and pentoxifylline led to a significant reduction of CD4+ cells in the distal parts of the grafts at all time points compared to untreated controls. This phenomenon was also observed in the proximal part of the grafts but only after 12 hours and after 7 days.
At 20 min after graft reperfusion only infliximab and etanercept significantly reduced CD8+ expression in the distal portion of the graft. However, at 12 hours and 7 days after reperfusion, all 3 forms of TNFa inhibition had led to a significant reduction of CD8+
expression in both, proximal and distal parts of the graft, compared to controls. Even after 6 months an anti-TNF- inhibition led to significantly reduced CD8+ cell expression in the proximal and distal part of the graft (except for etanercept in the proximal graft).
Of note, the number of infiltrating CD8+ cells was significantly increased in the distal parts of untreated transplant controls compared to native controls at all time points.
SDC, Figure 2
Immunohistology of ED1+ and CD25+ cells within proximal and distal grafts of all groups. There was a significantly reduced ED1 expression in both segments of the grafts of treated animals after 20 min (except for the proximal part of the graft under etanercept), 12 hours and 7 days, compared with untreated transplant controls. After 6 months only infliximab achieved a significant reduction of ED1+ expression.
The CD25+ expression in the proximal graft of treated animals was significantly reduced after 20 min, 12 hours and 7 days at all time points in the distal graft compared with untreated controls. Untreated transplant controls demonstrated a significant increase of CD25+ cells in the distal grafts at all time points compared with native controls, whereas the proximal part of the grafts showed a significant increase after 20 min, 12 hours and 7 days.
SDC, Figure 3
Immunohistology of Ox62+, MHCII+ and hsp70+ cells within proximal and distal grafts of all groups.
Only infliximab and pentoxifylline achieved a significant decrease of infiltrating OX62+
dendritic cells after 20 min and 7 days in the distal grafts compared with untreated controls. Nevertheless, the number of OX62+ DC tended to be reduced in all treatment groups.
The expression of MHC class II was hardly influenced by TNFa inhibition or the transplantation procedure.
The expression of Hsp 70 was significantly increased 20 minutes and 7 days post reperfusion following infliximab therapy in the proximal segment of the graft, as well as 7 days in the distal part, compared with native and transplanted controls.
SDC, Figure 4
A) Staining of MPO+ neutrophilic granulocytes in the lung sections of treated animals (anti-TNFa therapy with infliximab, etanercept, or pentoxifylline), in TX controls (without anti-TNFa therapy) and naive controls (neither treatment nor surgery) after orthotopic syngenic (LEW/LEW) small bowel transplantation. At 12 h post-reperfusion, the untreated controls displayed a significant increase in the amount of infiltrating neutrophils compared with the various treatment groups and naive controls. Treatment with etanercept and pentoxifylline led to significant reductions in neutrophil trafficking after 7 d compared with untreated controls. A notable tendency toward reduced infiltration of the lungs of treated recipients could still be observed after 6 mths.
B) MPO stains (light microscopy, magnification x400) of recipient lung tissues of untreated controls (left column) and infliximab-treated animals (right column) after 12 h (upper row), 7 d (middle row) and 6 mths (lower row). Note the massive infiltration of clusters of PMNs in the lungs of untreated animals. The pattern showed preference for neither the bronchioles nor the perivascular area. TNFa inhibition with infliximab mediated a significant decrease of PMNs at a level similar to that observed in naive controls.
SDC, Figure 5
Fluorescence activated cell sorting analysis (FACS): Detection of biotinylated infliximab (lower row) bound to membrane bound human (left column) and rat (right column) TNFa of mononuclear cells (MNC). Negative controls consisting of pure cell suspension of human MNC isolated from peripheral blood and rat MNC extracted from the spleen of LEW rats (upper row, left and right, respectively). The data representing the negative controls showed that no fluorescent phycoerythrin (PE) on MNC membranes was registered, indicating that no fluorescent-marked antibody did bind onto the cell surface.
Thus, all events are registered as negative events in the lower left quadrant. The results from the analyzed data of spleen suspensions of LEW rats (rat MNC + Infliximab +
Streptavidin- PE, lower row, right) and of the positive control (human MNC + Infliximab + Strepatavidin- PE, lower row, left) revealed a remarkable similarity (P>0.05) in the binding frequency of infliximab onto the cell surface. This shows that infliximab as an antibody directed against human TNFa binds to rat TNFa as well.
Table 1 CD8
proximal vs.
distal
Native control (N)
Tx control (C)
Infliximab (I)
Etanercept (E)
Pentoxi- phylline (P) after 20 minutes Ns p<0.001 p<0.01 p<0.01 p<0.001 after 12 hours Ns p<0.01 p<0.01 p<0.001 p<0.01 after 7 days Ns p<0.01 p<0.01 p<0.001 p<0.001 after 6 months Ns p<0.001 p<0.01 p<0.01 p<0.001 CD4
proximal vs.
distal
(N) (C) (I) (E) (P)
after 20 minutes Ns p<0.0001 ns p<0.05 p<0.0001 after 12 hours Ns p<0.0001 ns p<0.001 p<0.01 after 7 days Ns p<0.001 p<0.05 p<0.05 p<0.01 after 6 months ns p<0.001 p<0.01 p<0.01 p<0.001 CD25
proximal vs.
distal
(N) (C) (I) (E) (P)
after 20 minutes ns p<0.0001 ns p<0.05 ns after 12 hours ns ns p<0.01 p<0.01 p<0.05 after 7 days ns p<0.01 p<0.05 p<0.01 ns after 6 months ns p<0.001 p<0.01 p<0.05 ns ED1
proximal vs.
distal
(N) (C) (I) (E) (P)
after 20 minutes ns p<0.001 p<0.01 p<0.001 p<0.001 after 12 hours ns p<0.01 ns p<0.001 p<0.01
after 7 days ns p<0.001 ns p<0.01 ns
after 6 months ns ns p<0.05 p<0.01 p<0.05
Assessment of statistically significant differences (p-value) between proximal and distal graft segments for the expression of CD8+, CD4+, CD25+ and ED1+ in naive animals, untreated TX controls and after treatment with TNFa-inhibitors.
SDC, Figure 1
20min 12h 7d 6mths
0 2 4 6 8 10 12 14 16 18 20 22
CD8+ cells
vs.
all C
***
***
***
distal
0 1 2 3 4 5 6 7 8 9
CD8+ cells
20min 12h 7d 6mths
proximal
***
***
**
***
***
***
*
***
* ***
***
**
****** **
***
***
20min 12h 7d 6mths
0 5 10 15 20 25 30 35 40 45
CD4+ cells
proximal
*p<0.05 **p<0.01 ***p<0.001 native control (N)
Tx control (C)
Infliximab (I) Etanercept (E) Pentoxphylline (P)
20min 12h 7d 6mths
0 5 10 15 20 25 30 35 40 45 50 55 60
**
CD4+ cells
distal
**
* *
**
**** **
*****
***** *****
**
SDC, Figure 2
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6
CD25+ cells
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
CD25+ cells
20min 12h 7d 6mths 20min 12h 7d 6mths
distal
**
***
** ******
*** ***
***
***
***
*** * ***
***
*** ***
***
******
***
******
vs all C
***
*p<0.05 **p<0.01 ***p<0.001 native control (N)
Tx control (C)
Infliximab (I) Etanercept (E) Pentoxphylline (P)
proximal 0
5 10 15 20 25 30 35 40 45 50
**
***
**
***
ED1+ cells
proximal
20min 12h 7d 6mths 0
10 20 30 40 50 60 70
ED1+ cells
20min 12h 7d 6mths
vs all C
***
vs all C
***
distal
***
******
******
***
***
** ***
***
** ***
****** ***
SDC, Figure 3
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5
OX62+ cells
20min 12h 7d 6mths
distal
0 1 2 3 4 5 6 7 8 9 10
OX62+ cells
20min 12h 7d 6mths
vs N,I,P
***
proximal
* **
**
***
**
*
* *
**
**
*
**
*p<0.05 **p<0.01 ***p<0.001 native control (N)
Tx control (C) Infliximab (I) Etanercept (E) Pentoxphylline (P)
0 20 40 60 80 100 120 140
MHCII+ cells
proximal
20min 12h 7d 6mths 0
25 50 75 100 125 150
MHCII+ cells
distal
20min 12h 7d 6mths
** ***
***
**
*
**
**
***
*
***
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
hsp70+ cells
20min 12h 7d 6mths 0.0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
hsp70+ cells
proximal distal
20min 12h 7d 6mths
**
*** * *
***
**
*
***
*
SDC, Figure 4
12h 7d 6mths
0 25 50 75 100 125 150 175 200 225
MPO+ cells / quarter of field of view native control (N) Tx control (C)
Infliximab (I) Etanercept (E) Pentoxphylline (P)
**
***
**
*** **
**
Tx control Infliximab
12 h
7 d
6 mths A
B
SDC, Figure 5