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. 1998 Mar;66(3):1127-34.
doi: 10.1128/IAI.66.3.1127-1134.1998.

Salmonella flagellin induces tumor necrosis factor alpha in a human promonocytic cell line

F Ciacci-Woolwine  1 , I C Blomfield, S H Richardson, S B Mizel
Affiliations

Salmonella flagellin induces tumor necrosis factor alpha in a human promonocytic cell line

F Ciacci-Woolwine et al. Infect Immun. 1998 Mar.

Abstract

During infection of the gastrointestinal tract, salmonellae induce cytokine production and inflammatory responses which are believed to mediate tissue damage in the host. In a previous study, we reported that salmonellae possess the ability to stimulate tumor necrosis factor alpha (TNF-alpha) accumulation in primary human monocytes, as well as in the human promonocytic cell line U38. In this model system, cytokine upregulation is not due to lipopolysaccharide but is mediated by a released protein. In the present study, TnphoA transposon mutagenesis was used to identify the TNF-alpha-inducing factor. A mutant Salmonella strain which lacks the ability to induce TNF-alpha was isolated from a TnphoA library. Genetic analysis of this mutant demonstrated that the hns gene has been interrupted by transposon insertion. The hns gene product is a DNA-binding protein that regulates the expression of a variety of unrelated genes in salmonellae. One of the known targets of histone-like protein H1 is flhDC, the master operon which is absolutely required for flagellar expression. Analysis of other nonflagellated mutant Salmonella strains revealed a correlation between the ability to induce TNF-alpha and the expression of the phase 1 filament subunit protein FliC. Complementation experiments demonstrated that FliC is sufficient to restore the ability of nonflagellated mutant Salmonella strains to upregulate TNF-alpha, whereas the phase 2 protein FljB appears to complement to a lesser extent. In addition, Salmonella FliC can confer the TNF-alpha-inducing phenotype on Escherichia coli, which otherwise lacks the activity. Furthermore, assembly of FliC into complete flagellar structures may not be required for induction of TNF-alpha.

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Figures

FIG. 1
FIG. 1
Southern blot of FC32 chromosomal DNA. A 5-μg sample of DNA was digested with SalI and separated on a 0.5% agarose gel. The DNA was then transferred to a nylon membrane and probed with a 1,169-bp BglII fragment containing transposon TnphoA labeled with the Genius system as described in Materials and Methods. DNA bands were visualized by using the chemiluminescent substrate Lumi-Phos. The sizes of known fragments are shown on the right.
FIG. 2
FIG. 2
Effect of unpolymerized flagellin subunits on TNF-α activation in U38 cells. Serial dilutions of CM from SJW1103 or SJW2149 (a FliD− strain that fails to polymerize FliC into filaments) were tested on U38 cells as described in Materials and Methods. The amount of TNF-α in the control samples (8 ± 2 pg) has been subtracted from the values shown.
FIG. 3
FIG. 3
Effect of purified flagella on TNF-α expression in U38 cells and LPS-tolerant PBMC. Flagellar structures were isolated from Salmonella strain CD5 and tested on 3 × 106 U38 cells or PBMC at various concentrations. The curves shown are from two representative experiments. Control TNF-α levels were <1 pg/3 × 106 cells in unstimulated U38 cells and 7 ± 1 pg/3 × 106 cells in unstimulated PBMC. TNF-α levels in LPS-stimulated PBMC were 13 ± 4 pg/3 × 106 cells.
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