Lab 18: Tumor Necrosis Factor
The tumor necrosis factor (TNF) is a cytokine produced by myeloid cells stimulated with LPS. It can cause hemorrhagic necrosis of the tumor and is produced primarily by activated macrophages. It can also known to be produced by other cell types such as certain B lymphoblastoid cells, T lymphocytes, NK cells, mast cells, brain astrocytes and smooth muscle cells. The production of TNF can be induced by LPS, viruses, protozoa, mitogens, various cytokines, tumor cells, and phorbol esters. Once produced, can activate macrophages.
Two forms of TNF: Membrane-bound TNF (mTNF) and soluble form (sTNF)
Lymphotoxin (LT or TNF-beta) is similar in delayed-type hypersensitivity. It is produced by activated lymphocytes and some myeloma cells. Production induced by mitogens, phorbol ester, and IL-2. It inhibits B cells, kills T cells, activates macrophages, activates neutrophils, and kills fibroblasts and tumor cells.
TNF has pleiotropic effects such as:
and associated with
Release of TNF-alpha by macrophages induce local protective effects, but TNF-alpha can have damaging effects when released systemically.
Local effects: activates vascular endothelium and increases vascular permeability which leads to increased entry of IgG, complement, and cells and increased fluid drainage by lymph nodes; increased release of Ab, and migration of PMN leukocytes macrophages and lymphocytes.
Systemic effects: macrophages is insides blood vessel due to sepsis. fever, mobilization of metabolites, and shock, systemic release of TNF-alpha by activated macrophages, systemic edema, decreased blood volume, hypoproteinemina which all leads to disseminated intravascular coagulation, wasting, multiple organ failure, ultimately results in death.
In this lab we will determine the amount of cytolytic activity (cell killing) contained in a sample of recombinant TNF.
The cytolytic activity of any cytotoxic factor can be described in lytic units, where one lytic unit of TNF is the amount of TNF can lyse 50% of the target cells in a standardized cytolytic assay.
Change in procedure: Use MTT assay instead of chromium release assay.
Part A: preparation of targets, done by TA
Part B: omit since targets are not radiolabeled
Part C: Prepare TNF effector
Part D: change in procedure – omit step 1 and step 4. At step 3, after suspending cell pellet in 1 ml DMEM media, count live cells in hemocytometer without making dilution using trypan blue. Load into hemocytometer. Start from step 2 (page 70) for monlayer culture.
Part E: use MTT assay instead of 51Cr release assay. Add [appropriate] of TNF to each well as given in the lab manual.
SR = 50 uL of DMEM media A1-A3
0.001 U TNF/well = 50 of U/mL of TNF into wells A4-A6
0.1 U TNF/well = 50 of U/mL of TNF into wells A7-A9
1 U TNF/well = 50 of U/mL of TNF into wells A10-A12
10 U TNF/well = 50 of U/mL of TNF into wells B1- B3
100 U TNF/well = 50 of U/mL of TNF into wells B4- B6
Blank wells media only 100 ul into wells B10-B12
Incubate in a humidified incubator (37oC)with CO2 for 18 hours.
% killing = 100% - % cell survival
Formula for cell survival from the data:
Class Data (we are group one) for % killing:
|
Group: |
1 |
2 |
3 |
4 |
5 |
6 |
|
0.01 U |
17.4 |
3.9 |
1.1 |
8.5 |
4.3 |
24.8 |
|
0.1 U |
21.4 |
12.6 |
-- |
9.6 |
5.8 |
22.4 |
|
1.0 U |
33.3 |
53.9 |
5.1 |
13.6 |
20.5 |
27.7 |
|
10 U |
17.24 |
12.1 |
20.7 |
6.6 |
30.6 |
47.4 |
|
100 U |
48.7 |
62.0 |
59.1 |
12.6 |
46.8 |
85.7 |
Expected Results:
|
Unites of TNF: |
Cell percent survival: |
|
0.01 U |
0-10% |
|
0.1 U |
10-20% |
|
1.0 U |
20-30% |
|
10 U |
30-40% |
|
100 U |
40 + % |
Discussion:
In this lab we observed how the different concentration of TNF unites placed into assays with media and cells would affect the cell survival rate (percentage). The more TNF added, the higher the cell killing percentage (as seen in the data). TNF can be compared to the CTL lab in lab 18 where we varied the CTL concentration and saw that as the [CTL] increased, the more cells died off.
The trend of small to large percentage of killing was not always seen in the class data, as well as in our group data. Possible reasons was this was not enough TNF was added (i.e. concentration/dilution of TNF was done incorrectly). Not all of the six groups had the same degree of cell killing. Most likely each group used slightly different concentration of TNF. One group (group 3) had a negative killing rate (meaning cells actually proliferated, theoretically not possible in presence of TNF). It was unclear what had happened here.