Microbial Pathogenesis 1992 ; 13: 411-416

Short communications Suppression of LPS-inducible cytotoxicity in cytomegalovirus-infected THP-1 monocytic cell cultures does not correlate with a decrease in TNF-a antigen Lloyd W . Turtinen, David J . Tester and Thomas W . Flug Department of Biology, University of Wisconsin-Eau Claire, P .O. Box 4004, Eau Claire, WI 54702, U.S .A . (Received June 24, 1992 ; accepted in revised form August 3, 1992)

Turtinen, L . W . (Dept of Biology, University of Wisconsin-Eau Claire, P .O . Box 4004, Eau Claire, WI 54702, U .S .A .), D . J . Tester and T . W . Flug . Suppression of LPS-inducible cytotoxicity in cytomegalovirus-infected THP-1 monocytic cell cultures does not correlate with a decrease in TNF-a antigen . Microbial Pathogenesis 1992 ; 13 : 411-416 . We document suppression of tumor necrosis factor alpha (TN F-1) -associated cytotoxic activity in a human monocytic cell line (THP-1) infected with the mycoplasma free human cytomegalovirus (CMV) strain AD169 . Addition of lipopolysaccharide (LPS) to cell cultures that had been infected with CMV for 24 h resulted in a significant reduction in released cytotoxic activity to mouse L929 cells at 3-22 h post-LPS compared with mock-infected cultures . However, using an ELISA to measure TNF-a antigen levels in these culture supernatants, we found infected cultures had significantly higher TNF-a antigen levels than in mock-infected cultures following LPS induction . CMV alone also induced TNF-a release and possibly TNF-a inhibitor(s) which may have blocked TNF-a associated cytotoxic activity in CMV-infected THP-1 cell culture supernatants . Key words : human cytomegalovirus ; THP-1 cells; tumor necrosis factor ; monocytes ; cytotoxicity .

Human cytomegalovirus (CMV) infection has long been associated with disease in normal and immunocompromised individuals .' - ' Although CMV infection is most prevalent in immunocompromised AIDS' or tissue transplant patients, 5 .6 CMV itself induces a transient hyporesponsiveness in CMV-infected patients . 1-4 Attempts to define the mechanisms of immunosuppression by using in vitro models of immune function have succeeded in identifying some normal monocyte functions modulated by CMV infection . These include an impairment of monocyte antigen presentation to autologous lymphocytes' caused by a reduction in expression of MHC class II proteins, and a decrease in released interleukin-1 (IL-1)-associated proliferative activity," 4 presumably related to a decrease in IL-1 fl protein . In contrast, we reported earlier that more lipopolysaccharide (LPS)-inducible monokines, including TNF-a and IL-1fl proteins, were present in our CMV-infected THP-1 monocytic cell culture supernatants than in uninfected cultures ." To further investigate this apparent contradiction to the immunosuppressive properties of CMV, we measured both TNF-a antigen and 0882-4010/92/110411 +06 $08 .00/0

© 1992 Academic Press Limited



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associated cytotoxic activity released from THP-1 cultures . In addition, because the presence of mycoplasma in CMV stocks has been associated with increased immunosuppression in vitro, 1,14 we cultured CMV viral stocks to ensure no mycoplasma was present . Cell-free supernatants of the CMV strain AD1 69 were titered for extracellular virus using a black plaque ELISA, as previously described, 16 and contained 1 x10' plaque forming units (pfu)/ml . Cell-free viral supernatants were further tested for the presence of mycoplasma using two sensitive procedures . Samples were sent to the State Laboratory of Hygiene at the University of Wisconsin, Madison, WI, and cultured under both aerobic and anaerobic conditions using H-agar and diphasic E-broth . Replicate samples were sent to Quality Biotech Inc . (Camden, NJ) for testing and species identification using an immunoblotting assay . No mycoplasma could be cultured under aerobic or anaerobic conditions from the cell-free supernatants containing the CMV AD1 69 strain . Immunoblotting for detection and identification of the five most common cell culture contaminating mycoplasma was also negative . To measure the effect of CMV infection on LPS induction of cytotoxic activity from THP-1 monocytes, 5x10' cells were incubated with 1 ml of CMV stock or mock control for 3 h prior to washing and plating in a total volume of 10 ml for an additional 21 h . After 24 h, only 0 .02% of AD1 69 infected cells expressed CMV immediate early nuclear antigen using an immunoperoxidase staining procedure described previously . 15 Addition of LPS to a final concentration of 25,ug/ml at this time resulted in attachment of cells to the plastic dish, and a rapid release of cytotoxic activity into the culture supernatants (Table 1 ) . Cytotoxic activity in THP-1 cell supernatants was measured by using the standard cytotoxicity assay with antinomycin-treated L-292 cells ." L-929 cells were grown in 96-well flat-bottomed plates (3040 ; Falcon Plastics) at 40000 cells/ml in Eagle's minimum essential medium (EMEM) containing 10% endotoxin-free fetal calf serum (Hyclone Inc ., Logan, UT), glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin . Each well was incubated with a serially diluted test sample in the presence of 2 ug/ml actinomycin D in a humidified atmosphere at 37°C with 5% CO 2 . After 23 h, the test sample was removed, the plates washed with phosphate buffered saline (PBS), and cell lysis was detected by staining the plates with a 0 .25% solution of crystal violet in methanol/water (1 :4, v/v) . Cell monolayers exposed to growth medium were set at 0% lysis and those exposed to 3M guanidine hydrochloride for 1 h at 37 ° C were set at 100% lysis . One unit of cytotoxic activity was defined as the highest dilution of the sample that gave 50% cell lysis as measured on a micro-ELISA reader (Cambridge Instruments) set for absorption at 540 nm . Table 1 TNF-a and cytotoxicity levels in infected and uninfected THP-1 cell cultures following LPS induction Mock

Time' 0 3 22

TNF pg/ml 44+7° 2018±22 813+37

Cytotoxic units/ml ND° 156±5' 77+9

CMV AD169

pg/unit b

12 .9 10 .6

TNF pg/ml

Cytotoxic units/ml

pg/unit

112+23 2781+240 1820+470

ND 112±13 45+2

24 .5 40 .4

Hours post-LPS induction of THP-1 cells . • Picograms of TNF detected by ELISA divided by number of cytotoxic units in 1 ml of culture supernatant . c Average pg/ml of TNF±the standard error of the mean of three replicates . • ND = not detected within the sensitivity of cytotoxicity assay . • - = not calculated . Average cytotoxicity in units/ml ±the standard error of the mean of three replicates . a



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The 50% endpoint was determined by linear regression of a plot of the log reciprocal dilution against the absorbance at 540 nm . By 3 h post-LPS addition, mock-infected cultures contained approximately 156 units/ml as measured in a L929 cell cytotoxic assay . At 22 h post-LPS addition, 77 cytotoxic units/ml remained (Table 1) . Preinfection with CMV for 24 h significantly reduced (P < 0 .01) the amount of LPS-inducible cytotoxic activity present in THP-1 cell culture supernatants during the 3-22 h post-LPS induction period (Table 1 ) . In CMV AD169-infected cultures, cytotoxic activity averaged 112 units/ml at 3 h postLPS declining to 45 units/ml at 22 h post-LPS compared with mock controls . Because TNF-a is known to contribute to the majority of cytotoxic activity released from monocytes,'$ we used an ELISA to correlate TNF-a protein antigen levels with cytotoxic activity in culture supernatants, The level of TNF-a protein antigen released in THP-1 monocyte cultures was measured using a commercially available TNF-a ELISA kit (R&D Systems, Minneapolis, MN) . Preinfection with the CMV AD1 69 strain prior to LPS induction significantly increased (P < 0 .05) the amount of TNF-a present during 3 and 22 h post-LPS induction period compared with mock-infested levels [Table 1 and Fig . 1 (b)] . Infected culture fluids contained an average of 2781 pg/ml of TNF-a at 3 h post-LPS compared with 2018 pg/ml in mock-infected cultures . At 22 h post-LPS in CMV AD169-infected cultures, levels of TNF-a antigen remained over two-fold higher at 22 h post-LPS than in mock cultures [Table 1, Fig . 1 (b)] . We also looked at the ability of CMV stocks to induce TNF-a prior to the addition of L PS . T N F-a levels in culture supernatants were measured by ELISA at 2, 3, 16, 21, and 23 h after infection and prior to LPS addition [Fig . 1 (a)] . Mock-infected cultures showed a very small peak of 43 pg/ml TNF-a at 2 h post-infection . This was relatively

2000 (a)

40000

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35000

c

30000 25000 20000 15000

W. z H

10 000

Lhhh,

5000

nnn~n

-20

b..

-10

Hours before LPS addition

0

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Hours after LPS addition

Fig . 1 . The effect of CMV AD169 on the release of TNF-a from THP-1 myelomonocytic cell cultures . In (a) 5x106 THP-1 cells were infected with 1 ml of CMV strain AD169- •-, or uninfected mock control O-, for 3 h starting at ( - 24 h) prior to LPS addition . Following the 3 h infection, cells were washed with PBS, and expanded to 10 ml cultures for the remaining 21 h . In (b) E. co/i 055 :B5 LPS was added to a final concentration of 25 µg/ml after the 24 h infection . TNF-a levels were measured by ELISA before and after LPS addition . Each point represents the total pg of TNF-a in the culture supernatant obtained by multiplying the average pg/ml times the culture volume . Each point also shows the mean and standard error of the mean (vertical line) of three replicates . Points without vertical lines had standard errors too small to show . In (a) levels of TNF-a in AD169-infected cultures are statistically significantly higher (P < 0 .01) at 21 h before LPS addition than in mock supernatants . Likewise, in (b) levels of TNF-a in AD169-infected cultures were significantly higher (P < 0 .05) than in mock cultures during the 3-22 h post-induction period . Significance was determined using a Student's t-test .



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insignificant since the sensitivity of the TNF-a assay is between 7 .5 and 15 pg/ml in culture supernatants . In contrast, infection with both the CMV AD169 strain immediately induced TNF-a release into culture fluids with levels of TNF-a at 2 h postinfection 20-32-fold significantly higher (P

Suppression of LPS-inducible cytotoxicity in cytomegalovirus-infected THP-1 monocytic cell cultures does not correlate with a decrease in TNF-alpha antigen.

We document suppression of tumor necrosis factor alpha (TNF-alpha)-associated cytotoxic activity in a human monocytic cell line (THP-1) infected with ...
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