α v β 3 Integrin and Bacterial Lipopolysaccharide Are Involved in Coxiella burnetii -Stimulated Production of Tumor Necrosis Factor by Human Monocytes
Authors: D Raoult
J L Mege
R Toman
S M Hammami
J Dellacasagrande
C Capo
E Ghigo
Journal: Infection and Immunity (2000)
Discipline: IMMUNOLOGY
Keywords: Coxiella burnetii; Q fever; monocytes; TNF production; alpha(v)beta(3) integrin
Affiliations: <!--label omitted: 1-->Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, France,1 and, Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, France,1 and, <!--label omitted: 2-->Department of Rickettsiology and Chlamydiology, Institute of Virology, Slovak Academy of Sciences, 842 45 Bratislava, Slovak Republic2

Abstract:  Coxiella burnetii, the agent of Q fever, enters human monocytes through alpha(v)beta(3) integrin and survives inside host cells. In addition, C. burnetii stimulates the synthesis of inflammatory cytokines including tumor necrosis factor (TNF) by monocytes. We studied the role of the interaction of C. burnetii with THP-1 monocytes in TNF production. TNF transcripts and TNF release reached maximum values within 4 h. Almost all monocytes bound C. burnetii after 4 h, while the percentage of phagocytosing monocytes did not exceed 20%. Cytochalasin D, which prevented the uptake of C. burnetii without interfering with its binding, did not affect the expression of TNF mRNA. Thus, bacterial adherence, but not phagocytosis, is necessary for TNF production by monocytes. The monocyte alpha(v)beta(3) integrin was involved in TNF synthesis since peptides containing RGD sequences and blocking antibodies against alpha(v)beta(3) integrin inhibited TNF transcripts induced by C. burnetii. Nevertheless, the cross-linking of alpha(v)beta(3) integrin by specific antibodies was not sufficient to induce TNF synthesis. The signal delivered by C. burnetii was triggered by bacterial lipopolysaccharide (LPS). Polymyxin B inhibited the TNF production stimulated by C. burnetii, and soluble LPS isolated from C. burnetii largely mimicked viable bacteria. On the other hand, avirulent variants of C. burnetii induced TNF production through an increased binding to monocytes rather than through the potency of their LPS. We suggest that the adherence of C. burnetii to monocytes via alpha(v)beta(3) integrin enables surface LPS to stimulate TNF production in THP-1 monocytes. 

Retracted: No

PubPeer comments:
 Figure 1B. * Red boxes: an area under the bands appears to be visible two times. Such repetitive areas are not visible in other blot panels. ![file] , 
 Figure 2. * Cyan boxes: Two lanes in the TNF panel in A, one lane in the B panel and one lane in the C panel all look more similar than expected. * Yellow boxes:Two lanes in the B panel look more similar than expected ![file] , 
 Figure 3B. * Pink boxes: Similar to what is observed in Figure 1B, but unlike all the other blots, there appears to be a repetitive area under the bands in the TNF blot. ![file] , 
 As the 1st author of this study, I thank you for bringing these ~25-year old results to light! First, it is important to remember that at the time when these experiments were done, there was no digital camera and photo editing software we could have access to were very basic. Picture of gels shown in articles were often pictures of printouts from specific equipment (e.g. a gel imager), cut and pasted onto a sheet of paper on which legends were added. Such a page layout was then photographed with an analog camera... The digitalization of these documents was done by the journal editor. That said, let s go back to the data. First, the commented figures are not  blots . There are pictures of agarose gels stained with ethidium bromide and illuminated using UV light to show bands corresponding to fragments of cDNA amplified following RT-PCR. If the target mRNA is present in the sample, a single band is detected at a size depending on the selected pair of primers. It is a straight forward technique which rarely generates unwanted bands. I don t understand the concerns about figures 1B 3B: what would could have been hidden masked from these pictures? Regarding the repetitive patterns in figure 2, there are unlikely to be the result of duplications, backgrounds of boxed areas are different (see attached picture). Anyway, RT-PCR is only a semi-quantitative technique. In this article, the interpretation of the results weren t solely based on this technique. RT-PCR results in figures 1, 2 and 3 were all supported by quantitative techniques: ELISA for late time points and quantification of TNF transcripts for early time points using a specific kit (there was no quantitative PCR at that time). I hope the above details will bring you to the conclusion that the data presented in this article are an honest report of the actual experimental data which led us to conclude that u03b1vu03b23 was an important cell surface receptor in the immune response to C. burnetii. ![file] , 
 Dear Dr. Dellacasagrande, thank you for your reply, which is appreciated. Blot or not - I have run 1000s of ethidium-stained gels, and they somehow always looked like black gels with white bands; hence the confusion - the areas marked with colored boxes in my comments above still look more similar to each other that I expected. The similarities are not pixel-perfect, but more of the squint your eyes type. Here is a different version of the three panels from Figure 2, again, with boxes of the same color in shades of cyan blue and yellow orange pointing out the similarities. Green arrows focus attention to possible discontinuities in the background. ![file] , 
 #5 Could it be dust on the lens or in the camera that always produce the same pattern of black dots? Just wondering. Still for the differences in background transition from well to well the explanation may not hold. , 
 In 1998-2000 (at the time this manuscript was written) : - Digital cameras were indeed in their infancy but several models were already widely available even for non-professionals for example the popular coolpix 900 https: en.wikipedia.org wiki Nikon_Coolpix_series - Photo-editing softwares were already very widespread and not so basic. For example, in 1998 we had Photoshop 5.0 and in 2000 Photoshop 6.0. Slides and negatives scanners were also quite widespread. https: fr.wikipedia.org wiki Adobe_Photoshop#D%C3%A9veloppement The clone stamp tool was already present in photoshop version 5.0 https: www.youtube.com watch?v=JXqv2q2E3DU However, the similarities pointed here do not look like simple copy-paste with a clone stamp tool ( pixel-level similarities ) as can be seen in other more obviously manipulated images (but I m not a specialist). It is just that the argument that these technologies were not available at that time does not hold. ,