Main

Bacterial LPS (also known as endotoxin) as a constituent of the cell wall of Gram-negative bacteria is a major causative agent of septic shock. LPS starts a complex cascade of events in responsive cells, particularly in monocytes and macrophages, that leads to the production of endogenous mediators such as proinflammatory cytokines IL-1, tumor necrosis factor-α, IL-6, IL-8, and a number of other mediators. In Gram-positive bacteria, the major immunostimulatory components of the cell wall include peptidoglycan and lipoteichoic acid (1, 2).

A cell membrane component required for LPS-induced immunostimulation was recently identified to be a Tlr(3–5). According to the latest studies, Tlr2 and Tlr4 recognize different bacterial cell wall components. Tlr4 has been shown to mediate LPS-induced signal transduction (6, 7), whereas Tlr2 may mediate the response to yeast and Gram-positive bacteria (2, 8). Macrophages contain a surface protein called CD14, which binds ligands such as LPS (5, 7). However, CD14 does not participate directly in signaling. Rather, Tlr are essential for the innate immune response. Whereas the extracellular domain of Tlr, compatible with CD14, discriminates between pathogens, the cytoplasmic tail of Tlr triggers the cascade of intracellular mediators, leading to the activation of the nuclear factor κB and of the inflammatory response. Tlr are the mammalian homologues of the Drosophila Toll family that controls the dorsoventral patterning in the developing embryo and the antimicrobial response in the adult fly (9). So far, at least six of Tlr Drosophila have been identified in humans and mice (10). It has been proposed that Tlr control the switch from the innate to adaptive immune response (11). Tlr2 and Tlr4 initiate the transmembrane signaling that leads to activation of nuclear factor κB and induction of a number of inflammatory mediators (11, 12).

At present, there is little evidence of factors controlling the expression of Tlr in mammalian tissues (9, 10). The present study was undertaken to find out whether the expressions of Tlr2 and Tlr4 reveal trends during perinatal development and whether they are organ specific. The result is consistent with the possibility that the expression of Tlr controls the primary immune response to microbes. We propose that deficiencies in Tlr contribute to susceptibility to pulmonary infections during the perinatal period.

METHODS

The present study was approved by the Animal Experimentation Board of the University of Oulu.

Extraction of RNA.

Tissues were collected from mouse fetuses of different ages, known within ± 12 h. The age of the adults was 3 mo. The first day after conception was called d 0. The animals were killed by decapitation. The placenta, fetal lungs, and liver were frozen in liquid nitrogen. The tissues were ground in liquid nitrogen, and total RNA was isolated by Trizol reagent (GIBCO BRL, Life Technologies, Inc., Grand Island, NY, U.S.A.).

Quantitative reverse transcriptase-PCR analysis.

The absolute amounts of RNA were too small to be analyzed by Northern analysis. Half a microgram of the total RNA was used in each reverse transcriptase reaction. The quantitative PCR were driven by ABI PRISM 7700 Sequence Detection System (Perkin Elmer, Norwalk, CT, U.S.A.). The validation and reliability of this quantitative reverse transcriptase-PCR method has been reported (13). The primer sequences used for Tlr2 were 5′-GCCACCATTTCCACGGACT-3′ and 5′-GGCTTCCTCTTGGCCTGG-3′, and the TaqMan probe sequence was 5′(FAM)-TGGTACCTGAGAATGATGTGGGCGTG-(TAMRA)3′. The primer sequences used for Tlr4 were 5′-CCTCTGCCTTCACTACAGAGACTTT-3′ and 5′-TGTGGAAGCCTTCCTGGATG-3′, and the TaqMan probe sequence was 5′(FAM)-CCTGGTGTAGCCATTGCTGCCAACA-(TAMRA)3′. All results were normalized to 18S rRNA. The expression levels of Tlr2 and Tlr4 in each organ and in the whole body were related to each other. The whole body expression levels of Tlr2 and Tlr4 mRNA in 12-d fetus were very similar and valued as 1.0.

RESULTS

To study the ontogeny of Tlr2 and Tlr4 in mouse, the lungs, liver, and placenta were collected, and total RNA was isolated. The relative Tlr levels were quantified by reverse transcriptase-PCR. All results, normalized to 18S rRNA, were expressed as arbitrary units relative to the value 1.0 assigned to the whole fetus aged 12 d. The results showed very different expression patterns between tissues and between Tlr2 and Tlr4. In the lung, there was a several-fold increase in the Tlr2 and Tlr4 mRNA expression levels from a fetal age of 14–15 d to the term. In the adult lung, expression levels of Tlr2 and Tlr4 were higher than the expression levels in the newborn (Fig. 1). The regression lines for Tlr2 and Tlr4 were different from zero (p < 0.0001).

Figure 1
figure 1

Pulmonary expression levels of Tlr2 and Tlr4 in fetal, newborn, and adult mice. Four independent analyses were performed for each age group. Tlr mRNA levels are expressed as relative values; the whole body expression level of Tlr in 12-d-old fetus was valued as 1.0. NB indicates newborn; age < 20 h.

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In the liver, both Tlr2 and Tlr4 were prominent, and no developmental trends were detected (Fig. 2). In the placenta, on the other hand, the expression of Tlr4 was higher by 1 order of magnitude than that of Tlr2 (Fig. 3). The placental Tlr4 mRNA decreased during the second half of pregnancy (p < 0.01), whereas no significant trends in Tlr2 were evident.

Figure 2
figure 2

Hepatic expression levels of Tlr2 and Tlr4 in fetal, NB, and adult mice. Four or three (NB and adult) independent analyses were performed for each age group. Tlr mRNA levels are expressed as relative values; the whole body expression level of Tlr in 12-d-old fetus was valued as 1.0.

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Figure 3
figure 3

Expression levels of Tlr2 and Tlr4 in murine placenta. Four independent analyses were performed for each age group. Tlr mRNA levels are expressed as relative values; the whole body expression level of Tlr in 12-d-old fetus was valued as 1.0.

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DISCUSSION

In the present study, we have demonstrated for the first time that Tlr2 and Tlr4 mRNA expression is both tissue specific and dependent on the age. In the lung, the Tlr expression levels in immature fetuses were very low. Tlr increased 8-fold during the last trimester of murine pregnancy (from the late pseudoglandular to terminal sac stage) and further increased 2.5-fold after birth. The expression levels of Tlr2 and Tlr4 were similar. In contrast, the fetal liver showed 1 to 2 orders of magnitude higher Tlr mRNA expression levels than did the lung, and there were no remarkable changes in the expression during the fetal or postnatal life. In the placenta, the mRNA expression levels of Tlr4 during midpregnancy were higher by 1 order of magnitude than those of Tlr2.

Tlr2 and Tlr4 are expressed in macrophages (7, 14). Their expression levels influence the intensity of the inflammatory response to microbial toxins (6). The low levels of Tlr4 (1000 or fewer Tlr molecules per nonstimulated macrophage) (15) complicate the detection of Tlr in normal tissues by use of immunohistochemistry or in situ hybridization. Tlr expression levels were increased after the exposure to the LPS or the cytokines (16). At present, it is unknown whether the observed increase in Tlr in the lung tissue was due to the increase in the pulmonary content of macrophages, to the expression level of Tlr in the pulmonary macrophages, or to both. In the lung, the tissue concentration of macrophages tends to increase during prenatal development, whereas the number of alveolar macrophages increases rapidly after birth (17).

Whether the Tlr mRNA expression levels control the responsiveness of the innate immune system remains to be proven. It is of interest to note that some premature newborn infants are affected by hyperacute fulminant pneumonia due to Gram-positive (Group B Streptococcus, in particular) or Gram-negative (Escherichia coli and others) bacteria. In these cases, the initial acute phase response was deficient (18, 19). These data are supported by deficient responsiveness of the immature lung to LPS in vitro and an increase in the pulmonary LPS responsiveness toward term (20). Investigation of the genes and gene products responsible for deficient primary inflammatory response would help in the design of new strategies for prevention of life-threatening infections and inflammatory diseases.