On Day 4 participants had neutrophils isolated from 100 milliliters (mL) of acid-citrate dextrose (ACD)-anti-coagulated autologous venous blood and labeled in autologous plasma with up to 2.5 megaBecquerel (MBq) 111 Indium (111In)-tropolonate before being reinjected. Participants rested for 45 minutes (min) post-injection to allow for neutrophil equilibrium between the circulating and marginating neutrophil pools. Whole-body profiling was performed in a heavily shielded dedicated whole-body counter with 2 highly sensitive scintillation detectors with the recorded counts corrected for the physical decay of 111In to allow measurement of the effect of TCZ on the normal redistribution pattern of neutrophils and assessment of margination of neutrophils in the presence of TCZ. Distribution of radiolabelled neutrophils on Day 4 (45 min post re-injection) in the blood, liver/spleen and pelvic bone marrow, expressed as percentages of total body counts (TBCs).

Safety analysis population: Includes all the participants who received the single dose of randomized study medication. One participant in the polymorphonuclear leukocyte (PMN)-high group was excluded due to external contamination affecting profiling data.

On Day 4 participants had neutrophils isolated from 100 mL of ACD-anti-coagulated autologous venous blood and labeled with up to 2.5 MBq 111In-tropolonate before being reinjected. Participants rested for 45 min post-injection to allow for neutrophil equilibrium between the circulating and marginating neutrophil pools. Whole-body profiling was performed in a heavily shielded dedicated whole-body counter with 2 highly sensitive scintillation detectors with the recorded counts corrected for the physical decay of 111In to allow measurement of the effect of TCZ on the normal redistribution pattern of neutrophils and assessment of margination of neutrophils in the presence of TCZ. Distribution of radiolabelled neutrophils and peak counts, on Day 5 (24-hours post re-injection) in liver/spleen and pelvic bone marrow were decay corrected and expressed as percentages of Day 4 (45 minutes post re-injection).

Safety analysis population. One participant in the PMN-high group was excluded due to external contamination affecting profiling data.

On Day 4 participants had neutrophils isolated from 100 mL of ACD-anti-coagulated autologous venous blood and labeled with up to 2.5 MBq 111In-tropolonate before being reinjected. Participants rested for 45 min post-injection to allow for neutrophil equilibrium between the circulating and marginating neutrophil pools. Whole-body profiling was performed in a heavily shielded dedicated whole-body counter with 2 highly sensitive scintillation detectors with the recorded counts corrected for the physical decay of 111In to allow measurement of the effect of TCZ on the normal redistribution pattern of neutrophils and assessment of margination of neutrophils in the presence of TCZ. Distribution of radiolabelled neutrophils and peak counts, on Day 10 (6 days post re-injection) in liver/spleen and pelvic bone marrow were decay corrected and expressed as percentages of Day 4 (45 minutes post re-injection).

Safety analysis population. One participant in the PMN-high group was excluded due to external contamination affecting profiling data.

Neutrophil phagocytosis was assessed by flow cytometry using heat-killed Staphylococcal pneumonia (S.pneumonia) bacteria labeled with eFluor670. Phagocytosis was quantified by measuring the eFluor670 fluorescence from neutrophils containing phagocytosed bacteria. Experiments were performed using neutrophils (PMN) only, PMN plus S. pneumonia at 4 degrees(˚) centigrade (C) (to control for non-specific bacterial adherence to PMN cell surface), and PMN plus S. pneumonia at 37˚C. Change from baseline in the percentage of eFluor670+ neutrophils was calculated on Day 4.

Safety analysis population.

Neutrophil phagocytosis was assessed by flow cytometry using heat-killed Staphylococcal pneumonia bacteria labeled with eFluor670. Phagocytosis was quantified by measuring the eFluor670 fluorescence from neutrophils containing phagocytosed bacteria. Experiments were performed using neutrophils (PMN) only, PMN plus S. pneumonia at 4˚C (to control for non-specific bacterial adherence to PMN cell surface), and PMN plus S. pneumonia at 37˚C. Change from baseline in the eFluor670+ MFI was calculated on Day 4.

Safety analysis population

Neutrophils generate a respiratory burst using reactive oxygen species (ROS) to kill invading pathogens. When luminol is used as a substrate for ROS, a chemical reaction is produced resulting in photon emission (chemiluminescence) in primed and unprimed neutrophils following formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation which is quantifiable. fMLP stimulation of the respiratory burst is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in primed neutrophils. The maximal fMLP response is observed in primed neutrophils and is an ex vivo measure of the capacity of neutrophils to respond to pathogenic stimuli. In the current experiments, neutrophils were primed with tumor necrosis factor alpha (TNFα). Light emission was recorded on a luminometer. Absolute change from baseline in the production of ROS on Day 4 was reported.

Safety analysis population

Neutrophil apoptosis was measured using microscopy method with slides stained with Diff-Quik (modified Wright Giemsa stain) and morphology examined under oil immersion light microscopy with 100 times magnification. Neutrophils constitutively undergo apoptosis when cultured ex vivo, and this can be delayed by the addition of agents such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or TNFα. Apoptotic neutrophils were characterized with dark and pyknotic nuclei compared to the viable neutrophils. Change from baseline in the percentage of apoptotic neutrophils on Day 4 measured by microscopy is reported.

Safety analysis population

Ageing neutrophils translocate phosphatidylserine from the inner leaflet of the plasma membrane to the outer leaflet during the early stages of apoptosis. This translocation can be measured due to the affinity of Annexin V (AV) to bind exposed phosphatidylserine. Propidium Iodide (PI) is normally membrane-impermeable but enters cells in late apoptosis when their plasma membrane becomes leaky. Neutrophils constitutively undergo apoptosis when cultured ex vivo, and this can be delayed by the addition of agents such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or TNFα. Apoptosis was assessed by flow cytometry with fluorescein isocyanate-labeled recombinant human AV (AV-FITC) and PI staining and the change from baseline in the percentage of apoptotic neutrophils on Day 4 measured by flow cytometry is reported.

Safety analysis population

Neutrophil shape change is an indicator of the chemotactic ability of neutrophils to respond to and migrate to sites of inflammation. For determination of neutrophil shape change, fresh (0 min control), phosphate-buffered saline (PBS) control (30 min control) and formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated (30 min fMLP) PMNs (at 5 × 10^6 PMNs/ milliliter [mL]) were fixed with CellFIX (organic solvent used as fixative for adherent cells), 90 microliters (μL) transferred to each sample tube, and cold PBS added to stop further reaction. Shape change was assessed by measuring forward scatter (FSC) on flow cytometry. Change from baseline in the number of neutrophils with shape change on Day 4 was reported.

Safety analysis population

Neutrophil shape change is an indicator of the chemotactic ability of neutrophils to respond to and migrate to sites of inflammation. For determination of neutrophil shape change, fresh (0 min control), PBS control (30 min control) and fMLP-stimulated (30 min fMLP) PMNs (at 5 × 10^6 PMNs/ mL) were fixed with CellFIX, 90 μL transferred to each sample tube, and cold PBS added to stop further reaction. Shape change was assessed by measuring FSC on flow cytometry. Change from baseline in the percentage of neutrophils with shape change on Day 4 was reported.

Safety analysis population

Neutrophil shape change is an indicator of the chemotactic ability of neutrophils to respond to and migrate to sites of inflammation. For determination of neutrophil shape change, fresh (0 min control), PBS control (30 min control) and fMLP-stimulated (30 min fMLP) PMNs (at 5 × 10^6 PMNs/ mL) were fixed with CellFIX, 90 μL transferred to each sample tube, and cold PBS added to stop further reaction. Shape change was assessed by microscopy with neutrophils classified as shape-changed if they contained > 1 cell surface bleb or irregularity and change from baseline in percentage of neutrophil with shape change on Day 4 was reported.

Safety analysis population

Neutrophil surface receptor expression may be used to characterize the activation status of neutrophils. Fresh (0 min), PBS control (30 min) and fMLP-stimulated (30 min) PMNs (5 × 10^6 PMNs/mL) were fixed with CellFIX, and 90 μL transferred to each tube containing antibody mixture (2 μL cluster of differentiation [CD] 11b-brilliant violet (BV) 421, 2 μL CD16-FITC, 5 μL CD62L-allophycocyanin (APC) and 5 μL CD162-phycoerythrin [PE]) or isotype control mixture of equivalent volumes. After 30 minutes of incubation on ice and in the dark, cold PBS was added to stop further reaction. Surface marker expressions were quantified by flow cytometry.

Safety analysis population