Roles of Islet Toll-Like Receptors in Pig to Mouse Islet Xenotransplantation

Animals

Porcine pancreases were harvested from adult female farm pigs at local slaughterhouses. Mice deficient in myeloid differentiation primary response 88 (MyD88) and TLR4 were the gift of Shizo Akira (Osaka University, Osaka, Japan). Male mice at 8–12 weeks of age were used as transplant recipients. The animal use protocol of this study was approved by the Institutional Animal Care and Use Committee of Seoul National University and Seoul National University Hospital.

Islet Isolation

Isolation and purification of porcine islets were performed as previously described (8), with minimal modifications. Briefly, the harvested pancreas was distended intraductally with University of Wisconsin solution containing Liberase DL (Roche Biochemicals, Basel, Switzerland) at a concentration of 2 ml/g of pancreatic tissue. The distended pancreas was digested in a modified Ricordi chamber (Biorep Technologies, Miami, FL, USA), and liberated islets were separated using density gradient on a Cobe 2991 cell separator (Gambro BCT Inc., Lakewood, CO, USA).

In Vitro TLR Stimulation

Porcine islets [1,000 islet equivalent (IEQ)] were grown in M199 media (Gibco) with 10% adult porcine serum (Gibco) and 1% penicillin/streptomycin solution (Gibco). Porcine islets were stimulated with polyinosinic/polycytidylic acid (poly I:C, 30 μg/ml, Sigma-Aldrich, MO, USA) or Escherichia coli 0111:B4 lipopolysaccharide (LPS, 0.5–1 μg/ml, Sigma-Aldrich) for 6–72 h.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from islets using an RNA mini kit (Invitrogen, Carlsbad, CA, USA). The total RNA (1 μg) was reverse transcripted to produce cDNA using Superscript III reverse transcriptase (Invitrogen). Amplification was carried out using the PCR Thermal Cycler Dice^™ (TaKaRa, Shiga, Japan). The β-actin gene was used for the quantitative titration of cDNA samples, and gene expression of target genes was presented as their expression relative to expression of β-actin gene. Groups without LPS/poly I:C stimulation were regarded as control groups.

After pig to mouse islet transplantation, frozen islet tissue was homogenized and grounded up by mortar and pestle. After RNA extraction, tissue expression of both murine and porcine chemokines/cytokines was analyzed using RT-PCR and was presented as relative expression normalized to expression of β-actin gene. Groups without LPS stimulation were regarded as control groups. We measured expression of chemokine C-X-C motif ligand 1 (CXCL1) as a functional homologue of interleukin-8 (IL-8) in mice, where there is no direct homologue of IL-8.

Primer sequences are available upon request.

Enzyme-Linked Immunosorbent Assay (ELISA) and Flow Cytometry

Protein levels of secreted chemokines in the culture supernatants of porcine islets were measured using cross-reactive human chemokine ELISA kits (RayBiotech Inc., Norcross, GA, USA).

For flow cytometric analysis, porcine islets were dissociated into single-cell suspensions by gentle agitation in accutase (Gibco, Gaithersburg, MD, USA). Islet cells were stained with anti-vascular cell adhesion molecule-1 (VCAM-1) or anti-intercellular adhesion molecule-1 (ICAM-1) antibodies generated in-house (35) and then stained with FITC-conjugated secondary antibodies (rabbit IgG, Invitrogen, Carlsbad, CA, USA). The stained cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Transmigration Assay

Human peripheral blood was taken from healthy donors (n = 3; age 32.3 ± 3.2 years; male/female, 2:1) after informed consent and ethical approval at Seoul National University Hospital. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by centrifugation using Ficoll-Plaque PLUS (GE Healthcare Life Science, Uppsala, Sweden). PBMCs (3×10⁵)were added to the top of the insert in the Neuro Probe 48-Well Micro Chemotaxis Chamber (Neuro Probe, Inc., Gaithersburg, MD, USA). Culture supernatants (30 μl) were added below the culture insert. The transmigration assay was conducted for 2.5 h at 37°C. The number of transmigrated cells was calculated by counting the number of stained cells. Human PBMCs were pretreated with anti-RANTES (regulated on activation normal T-cell expressed and secreted) antibodies (4 μg/ml, R&D Systems, Minneapolis, MN, USA) or isotype control goat IgG (4 μg/ml, Sigma-Aldrich) for 30 min before transmigration assays in order to confirm the chemotactic effect of porcine RANTES.

Chromogenic Factor Xa Generation Assay and Thrombin Generation Assay

Tissue factor activity was analyzed by chromogenic factor Xa generation assay. Briefly, islets were incubated with activated factor VIIa (500 ng/ml; American Diagnostica, Stamford, CT, USA) and factor X (1 unit/ml; American Diagnostica) for 45 min at 37°C. The supernatant containing factor Xa was transferred to a new well and incubated with the chromogenic substrate S-2765 (DiaPharma, West Chester, OH, USA). The 96-well plates (BD, Franklin Lakes, NJ, USA) were read in an automated plate reader (Bio-Tek Instruments Inc., Winooski, VT, USA) at 405 nm for 10 min.

In order to measure the activity of Fgl-2 (fibrinogen-like protein 2), a thrombin generation assay was performed as described previously (19). Briefly, islets (500 IEQ) were incubated with an equal volume of human prothrombin (American Diagnostica) for 20 min at 37°C. After centrifugation, the supernatant was transferred to a new well, where Chromozym TH^™ (Roche Biochemicals), a chromogenic substrate of human thrombin, was added. OD₄₀₅ was measured using an automated plate reader.

Glucose-Stimulated Insulin Secretion Test

Porcine islets were incubated with poly I:C (30 μg/ml) or LPS (1 μg/ml) overnight (2.8 mM glucose). Insulin secretion was stimulated by treatment of cells with 20 mM glucose (Sigma-Aldrich) for 1 h at 37°C. Next, insulin concentration in the supernatant was determined by radio-immunoassay (Biosource, Nivelles, Belgium). The stimulation index was calculated as the ratio of stimulated (20 mM glucose) to basal (2.8 mM glucose) insulin release.

MyD88 Knockdown for the Porcine Islets Using shRNA Lentivirus

A shRNA sequence against porcine MyD88 mRNA (5′-GAGGACTGCCGAAAGTATA-3′) or the control sequence without any knockdown activity (5′-CTGCCCC AGCGATATCCAG-3′) was selected following a previous study (4) and was inserted into the expression vector, pDSL_ hpUGIP containing GFP (ATCC, Manassas, VA, USA). HEK-293T cells (ATCC) were transfected with the expression vectors together with packaging vectors (pMDLg/pRRE; pRSV-Rev, Addgene Inc., Cambridge, MA, USA) and enveloping vectors (pCMV-VSV-G, Addgene Inc.) by the calcium phosphate method for generation of shRNA lentivirus vector. The virus-containing supernatant was collected after 48 h and concentrated approximately 20-fold using Centricon Plus-70 (Millipore, Billerica, MA, USA). Porcine islets were infected with lentivirus [20 multiplicity of infection (MOI)] with the shMyD88 or control sequence for 8 h. After washing and an additional 16 h of culture, porcine islets were stimulated by LPS (1 μg/ml) for 6–48 h. The degree of MyD88 suppression was measured using both RT-PCR and Western blot (anti-MyD88 antibody, Abcam Inc., Cambridge, MA, USA). MyD88 knockdown-mediated suppression of LPS-induced inflammatory mediators and procoagulants was analyzed by RT-PCR.

Pig Islet to Mice Transplantation

MyD88 or TLR4 knockout C57BL6/J mice were rendered diabetic using a single intraperitoneal injection of streptozotocin (200 mg/kg, Sigma). Porcine islets (5,000–10,000 IEQ) were transplanted under the renal capsule of the recipients. Mice were treated intraperitoneally with anti-cluster of differentiation 154 (CD154) antibodies (0.5 μg, MR-1, BioXell, Lebanon, NH, USA) on days 0, 2, and 4 after transplantation. LPS (100 μg) was intraperitoneally administered on days 3, 5, and 7. Blood glucose concentration was measured using Accu-Chek^® Performa^® (Roche Diagnostics, Mannheim, Germany). Graft rejection was defined as the occurrence of a blood glucose concentration >350 mg/dl on two consecutive tests. Posttransplant normoglycemia was defined as random blood glucose concentration less than 200 mg/dl. In the case of islet recipients that were persistently normoglycemic by the end of the observation period (>200 days), the graft function was confirmed by unilateral nephrectomy of the kidney bearing the graft and documentation of the reappearance of diabetes.

Histological Analysis

Mice were sacrificed on day 8 after porcine islets were transplanted to TLR4 knockout C57BL6/J mice, and the kidneys with islet grafts were harvested. Grafts were embedded in Tissue-Tek^® optimal cutting temperature (OCT) embedding compound (Sakura Finetek, Tokyo, Japan) and snap-frozen in liquid nitrogen for storage at −80°C. Guinea pig anti-insulin antibodies (DAKO, Carpinteria, CA, USA) and rat anti-mouse epidermal growth factor (EGF)-like module-containing mucin-like hormone receptor-like 1 (F4/80), rat anti-mouse CD4, rat anti-mouse CD8, and rat anti-mouse protein tyrosine phosphatase, receptor type C (CD45, B220; all e-Bioscience, San Diego, CA, USA) were used at 4°C overnight as the primary antibodies in immunofluorescent staining. Next, sections were stained with Alexa 488 goat anti-guinea pig IgG and Alexa 594 rabbit anti-rat IgG (Invitrogen) at room temperature for 1 h. Cell nuclei were counterstained with DAPI (Sigma-Aldrich) for 10 min. Cellular infiltration into the grafts was assessed using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Inc., Thornwood, NY, USA).

Statistical Analysis

Most values were expressed as mean ± standard deviation. All analyses were performed using SPSS statistical software (version 17.0; Armonk, NY, USA). Continuous variables were compared using a t test, and categorical variables were compared using Fisher's exact test. Comparison between Kaplan–Meier survival curves was performed using the log-rank test. Values of p < 0.05 were regarded as statistically significant. For multiple comparison, values of p < 0.025 were considered statistically significant after Bonferroni adjustments for multiple comparisons.

In Vitro TLR Stimulation in Porcine Islets Induced Expression of Proinflammatory Chemokines and Cytokines

Porcine islets expressed TLRs (TLR1–10) at resting status in RT-PCR (Fig. 1A). Both poly I:C and LPS stimulation induced RANTES (Fig. 1B), MCP-1 (Fig. 1C), interferon γ-induced protein 10 (IP-10) (Fig. 1D), and interleukin-8 (IL-8) (Fig. 1E) mRNA in porcine islets. Expression of interleukin-6 (IL-6) (Fig. 1F), interferon-α (IFN-α) (Fig. 1G), and interferon-β (IFN-β) (Fig. 1H) was also induced in porcine islets by poly I:C and LPS stimulation. Secretion of RANTES (Fig. 2A) and MCP-1 (Fig. 2B) as determined by the human chemokine ELISA kits was also increased in parallel with RT-PCR data. Overall, these results suggested that innate stimuli can stimulate TLR3 and TLR4 in porcine islets and thereby induce a proinflammatory response from porcine islets themselves.

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Figure 1.

TLR stimulation in porcine islets induced mRNA expression of proinflammatory chemokines and cytokines. (A) Toll-like receptor (TLR) expression in porcine islets at resting status. The expressions of TLR in porcine islets after overnight culture were analyzed by semiquantitative RT-PCR. (B–H) Induction of regulated on activation normal T-cell expressed and secreted (RANTES) (B), monocyte chemoattractant protein-1 (MCP-1) (C), interferon (IFN) γ-induced protein 10 (IP-10) (D), interleukin-8 (IL-8) (E), IL-6 (F), IFN-α (G), and IFN-β (H) in porcine islets in response to TLR stimulation. Expression of chemokines and cytokines after stimulation of polyinosinic/polycytidylic acid (poly I:C; 30 μg/ml) or lipopolysaccharide (LPS; 1 μg/ml) were analyzed by semiquantitative RT-PCR. The β-actin gene was used for the quantitative titration of cDNA samples, and gene expression of target genes was presented as their expression relative to expression of β-actin gene. Groups without LPS/poly I:C stimulation were regarded as control groups. *,#Significant differences (p < 0.01, p < 0.025, respectively) in comparison to control values at each time point. Each bar indicates the mean and standard deviation among triplicates. These results are representative of at least three independent experiments.

Porcine Islet Supernatants Stimulated by TLR Agonists Induced Chemotaxis of Human Leukocytes Across the Species Barrier

Islet supernatants cultured with poly I:C or LPS induced chemotaxis of human peripheral blood mononuclear cells (Fig. 2C). When transmigration assays were performed after pretreatment with anti-RANTES blocking antibodies, the chemotactic effects of the islet supernatant were significantly attenuated (p < 0.01), whereas isotype control goat IgG did not affect the chemotactic activity. These data indicated that porcine chemokines, especially RANTES, secreted from islets in response to TLR stimulation can recruit human leukocytes across the species barrier.

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Figure 2.

TLR stimulation in porcine islets induced secretion of porcine chemokines and chemotactic activity toward human leukocytes and upregulated expression of adhesion molecules. (A, B) Secretion of chemokines from porcine islets in response to TLR stimulation. After porcine islets were cultured with poly I:C (30 μg/ml) or LPS (1 μg/ml) for 48–72 h, concentrations of RANTES (A) and MCP-1 (B) in the supernatants were measured by ELISA. *Significant differences (p < 0.01) in comparison to basal values. (C) Chemotactic activities of supernatants from porcine islets stimulated by poly I:C or LPS toward human peripheral blood mononuclear cells. Porcine islets were cultured with poly I:C (30 μg/ml) or LPS (1 μg/ml) for 48 h, and the culture supernatants were used in transmigration assays. Anti-RANTES blocking antibodies decreased chemotactic activity significantly in transmigration assays (p < 0.01), whereas isotype control goat IgG did not affect the chemotactic activity. *Significant differences (p < 0.01) in comparison to the unstimulated control group. **Significant differences (p < 0.01) between the TLR stimulation groups and the anti-RANTES blocking groups. (D–E) Upregulation of vascular cell adhesion molecule-1 (VCAM-1) (D) and intercellular adhesion molecule-1 (ICAM-1) (E) in porcine islets by TLR stimulation. Porcine islets were cultured with poly I:C (30 μg/ml) or LPS (1 μg/ml) for 48 h. Surface expression of VCAM-1 and ICAM-1 in porcine islets was analyzed by flow cytometric analysis. Each bar indicates the mean with a standard deviation among triplicates. These results were representative of two to three independent experiments.

TLR Stimulation in Porcine Islets Induced Expression of Adhesion Molecules

Next, induction of adhesion molecules in porcine islets in response to TLR stimulation was investigated. Stimulation with either poly I:C or LPS increased expression of both VCAM-1 (p = 0.02 for poly I:C, p = 0.08 for LPS) (Fig. 2D) and ICAM-1 (p < 0.01 for poly I:C, p = 0.09 for LPS) (Fig. 2E) in porcine islets.

TLR Stimulation in Porcine Islets Induced Procoagulant Activation

Both poly I:C and LPS stimulation in porcine islets induced mRNA expression of tissue factor (Fig. 3A) and Fgl-2 (Fig. 3B). When functional activity of tissue factor was measured, tissue factor activity increased in parallel with RT-PCR data after stimulation by either poly I:C or LPS (Fig. 3C). Functional activities of Fgl-2 also increased in response to TLR stimulation in the thrombin generation assay (Fig. 3D). Procoagulant induction in porcine islets was more prominent in poly I:C stimulation than in LPS stimulation. Taken together, these data showed that TLR stimulation in porcine islets induced procoagulants as well as proinflammatory mediators.

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Figure 3.

TLR stimulation in porcine islets induced procoagulant activation. Porcine islets were cultured with poly I:C (30 μg/ml) or LPS (1 μg/ml) for 6–24 h. (A, B) Expression of tissue factor (A) and fibrinogen-like protein 2 (Fgl-2) (B) in porcine islets after coculture with poly I:C or LPS were analyzed by semiquantitative RT-PCR. The β-actin gene was used for the quantitative titration of cDNA samples, and gene expression of target genes was presented as their expression relative to expression of β-actin gene. Groups without LPS/poly I:C stimulation were regarded as control groups. *,#Significant differences (p < 0.01, p < 0.025, respectively) in comparison to unstimulated controls at each time point. (C, D) Functional activity of tissue factor (C) and Fgl-2 (D) in porcine islets by TLR stimulation was measured using a chromogenic factor Xa generation assay (C) and thrombin generation assay (D), respectively. *,#Significant differences (p < 0.01, p < 0.025, respectively) in comparison to unstimulated controls at each time point. Each bar indicates the mean and standard deviation among triplicates. The results were representative of three independent experiments.

TLR Stimulation Did Not Influence Insulin Secretion From Porcine Islets

When impact of TLR stimulation on insulin secretion was evaluated using a glucose stimulated insulin secretion test, stimulation with neither poly I:C nor LPS significantly influenced insulin secretary function, a parenchymal function of islets (p = 0.16 for poly I:C; p = 0.78 for LPS) (Fig. 4).

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Figure 4.

TLR stimulation in porcine islets did not affect insulin secretion from islets. When the impact of TLR stimulation on insulin secretion was evaluated using a glucose stimulated insulin secretion test, stimulation with neither poly I:C nor LPS influenced insulin secretion significantly (p = 0.78 for poly I:C; p = 0.16 for LPS). Each bar indicates the mean and standard deviation among triplicates. The results were representative of three independent experiments.

MyD88 Knockdown Suppressed the LPS-Induced Proinflammatory and Procoagulant Response of Porcine Islets

Infection of porcine MyD88-shRNA lentivirus (20 MOI) significantly attenuated expression of MyD88 mRNA in porcine islets in semiquantitative RT-PCR (p < 0.01) (Fig. 5A). The knockdown effect in protein expression of porcine MyD88 was about 80–90% according to the Western blot (p < 0.01) (Fig. 5B).

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Figure 5.

Knockdown of porcine MyD88 suppressed the LPS-mediated induction of chemokines and procoagulants in porcine islets. (A, B) Lentiviral shRNA knocked down expression of porcine myeloid differentiation primary response 88 (MyD88) in islets successfully. Porcine islets were transduced with 20 multiplicity of infection (MOI) of lentivirus (with a control or shMyD88 sequence). Expression of MyD88 in lentivirus-transduced, porcine islets with LPS (1 μg/ml) stimulation was analyzed by both semiquantitative RT-PCR (A) and Western blot (B). The β-actin was used for the quantitative titration of cDNA samples and as a quantity control in western blot analysis. Expression of MyD88 gene and protein was presented as their expression relative to expression of β-actin. (C–F) Knockdown of porcine MyD88 in islets attenuated LPS-induced expression of chemokines and cytokines significantly. shMyD88 lentivirus-transduced islets were cultured with LPS (1 μg/ml) for 24–48 h. Expression of chemokines (RANTES, MCP-1, IP-10) and IL-6 in porcine islets was analyzed by semiquantitative RT-PCR. The β-actin gene was used for the quantitative titration of cDNA samples. (G, H) Knockdown of porcine MyD88 in islets attenuated procoagulant induction by LPS stimulation significantly. shMyD88 lentivirus-transduced islets were cultured with LPS (1 μg/ml) for 6–24 h. Expression of tissue factor (G) and Fgl-2 (H) in porcine islets after lentivirus infection was analyzed by both semiquantitative RT-PCR. The β-actin gene was used for the quantitative titration of cDNA samples. Gene expression of target genes was presented as their expression relative to expression of β-actin gene. *,#Significant differences (p < 0.01, p < 0.05, respectively) in comparison to controls at each time point. Each value indicates the mean and standard deviation among triplicates. The results were representative of three independent experiments.

MyD88 knockdown suppressed LPS-mediated induction of chemokines (RANTES, MCP-1, IP-10) and cytokines (IL-6) in porcine islets (p < 0.05) (Fig. 5C–F). LPS-mediated induction of tissue factor and Fgl-2 from porcine islets was also suppressed by MyD88 knockdown in both RT-PCR (p < 0.05) (Fig. 5G–H). These data supported that TLRs in porcine islets played a significant role in induction of proinflammatory and procoagulant responses and had a functional significance.

TLR4 Stimulation in Islets Led to Xenograft Rejection

The impact of in vivo TLR stimulation in porcine islets while minimizing TLR stimulation on the recipient mice was investigated using MyD88 or TLR4 knockout mice. Porcine islets were transplanted into diabetic MyD88 or TLR4 knockout mice (Fig. 6A). Anti-CD154 treatment prolonged porcine islet xenograft survival with a median survival time (MST) longer than 200 days in both MyD88 knockout and TLR4 knockout recipients. LPS stimulation led to xenograft rejection in both MyD88 knockout (MST, 15 days for LPS group; LPS group vs. MR-1 group, p = 0.02) (Fig. 6B) and TLR4 knockout recipients (MST, 62.5 days for LPS group; LPS group vs. MR-1 group, p < 0.05) (Fig. 6C). Overall, LPS stimulation, mainly in porcine islets, interfered with islet xenograft tolerance induction by costimulatory blockade. These data suggested that TLR activation in donor islets plays a significant role in islet xenograft rejection.

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Figure 6.

TLR stimulation in porcine islets interfered with tolerance induction in pig to mice islet transplantation. (A) Porcine islets were transplanted to MyD88 knockout or TLR4 knockout mice. Mice were treated with anti-cluster of differentiation 154 (CD154) antibodies (MR-1) on days 0, 2, and 4 after transplantation. LPS was intraperitoneally administered to the experimental group on days 3, 5, and 7 after transplantation. (B, C) Anti-CD154 antibody (MR-1) treatment without LPS induced islet xenograft tolerance in more than half of MyD88 knockout (B) or TLR4 knockout (C) recipients. However, LPS stimulation in porcine islets led to xenograft rejection in both MyD88 knockout recipients (median survival time, 15 days for LPS group; LPS group vs. MR-1 group, p = 0.02, log-rank test) (B) and TLR4 knockout recipients (median survival time, 62.5 days for LPS group; LPS group vs. MR-1 group, p < 0.05, log-rank test) (C).

In Vivo TLR4 Stimulation Increased Proinflammatory Chemokines and Cytokines in Islet Xenograft

Consistent with in vitro data, expression of porcine chemokines (RANTES, MCP-1, IL-8) and cytokines (IL-6, IFN-α, IFN-β) in islet grafts was significantly increased by stimulation of LPS compared with the control group (p < 0.05) (Fig. 7A). Expression of murine chemokines (RANTES, MCP-1, IP-10) and cytokines (IL-6, TNF-α) from the recipients was also increased secondarily to LPS stimulation (p < 0.05) (Fig. 7B), whereas there was no significant difference in the expression of IFN-γ, IL-4, or IL-10 (Fig. 7B).

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Figure 7.

TLR stimulation in porcine islets increased the expression of proinflammatory chemokines and cytokines in both donors and recipients in pig to mouse islet transplantation. After porcine islets were transplanted to TLR4 knockout C57BL6/J mice, anti-CD154 antibodies were administered with or without LPS and islet xenografts were harvested on day 8. Tissue expression of both porcine (A, RANTES, MCP-1, IP-10, IL-8, IL-6, IFN-α, and IFN-β) and murine [B, RANTES, MCP-1, IP-10, chemokine C-X-C motif ligand 1 (CXCL1), IL-6, tumor necrosis factor (TNF)-α, IFN-γ, IL-4, and IL-10] chemokines/cytokines were analyzed using semiquantitative RT-PCR. The β-actin gene was used for the quantitative titration of cDNA samples, and gene expression of target genes was presented as their expression relative to expression of β-actin gene. Groups without LPS stimulation were regarded as control groups. *,#Significant differences (p < 0.01, p < 0.05, respectively) as compared with the control group without LPS. Each value indicates the mean and standard deviation among triplicates. The results were representative of two independent experiments.

In Vivo TLR4 Stimulation Increased Infiltration of Inflammatory Cells Into Islet Xenograft

When inflammatory cell infiltration around the islet xenograft was analyzed on day 8 after transplantation using confocal microscopy (Fig. 8), LPS stimulation increased infiltration of both innate (macrophages, p = 0.02) (Fig. 8A, B, F) and adaptive (CD4⁺ T-cells, p < 0.01) (Fig. 8C, F) (CD8⁺ T-cells, p = 0.03) (Fig. 8D, F) (B-cells, p < 0.01) (Fig. 8E, F) immune cells in parallel with higher expression of inflammatory chemokines by LPS stimulation.

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Figure 8.

TLR stimulation in porcine islets increased inflammatory cell infiltration into islet xenografts in pig to mouse islet transplantation. After porcine islets were transplanted to TLR4 knockout C57BL6/J mice, anti-CD154 antibodies were administered with or without LPS and islet xenografts were harvested on day 8. (A–F) When infiltration of macrophages (A, B), CD4⁺ T-cells (C), CD8⁺ T-cells (D), and B-cells (E) was analyzed using confocal microscopy, LPS stimulation increased the infiltration of all sets of inflammatory cells around islet grafts compared with the control group. Green indicates insulin staining, blue indicates DAPI staining, and red indicates staining for specific cell surface markers {F4/80 [epidermal growth factor (EGF)-like module-containing mucin-like hormone receptor-like 1] for macrophages, CD4, CD8, and B220 [protein tyrosine phosphatase, receptor type, C; CD45] for B-cells}. Original magnification: 400×. (F) Quantitative analysis of infiltrating cells into islet xenografts. Numbers of graft-infiltrating cells were counted and averaged in three high-power fields (400×) per each mouse. When the numbers of infiltrating cells were compared, infiltration of macrophages, T-cells, and B-cells increased significantly in the LPS group compared with the control group (*p < 0.01, #p < 0.05). Each value indicates the mean and standard error among three mice. The results were representative of three independent experiments.