The Innate Immune Response Differs in Primary and Secondary Salmonella Infection

Mice

C57BL/6 mice were used between 7 and 10 wk of age. Age-matched naive controls were included as stated. All mice were bred and housed in the animal facilities at Lund University and were provided with food and water ad libitum.

Bacteria and immunization procedures

S. typhimurium χ4550 and χ4666 ΔasdA1 derivatives carrying the asd⁺ plasmid pYA3259 encoding OVA were used in these studies (32, 33). χ4550-OVA is attenuated (Δcya-1 Δcrp-1 ΔasdA1) and has a 50% lethal dose of >10¹⁰ bacteria by the oral route. χ4666-OVA (ΔasdA1) has a 50% lethal dose of <10⁵ bacteria by the oral route, with doses from 10⁵ up to 10¹⁰ χ4666-OVA resulting in death on days 7–10 in naive animals (our unpublished observations). Both strains are SR11 derivatives. Overnight cultures of bacteria were grown in Luria-Bertoni broth and incubated at 37°C with shaking. The concentration of bacteria was determined spectrophotometrically, and bacteria were washed, resuspended at the appropriate concentration in sterile PBS, and used to infect mice. The actual bacterial doses administered were determined by counting colonies following plating of serial dilutions of the inoculum on Luria-Bertoni agar plates.

To examine the innate response to primary Salmonella infection, a single oral dose of χ4666 was used. For oral immunizations, mice were treated intragastrically with 0.1 ml 1% sodium bicarbonate, followed 5–10 min later by intragastric inoculation of bacteria in a volume of 0.2 ml. The oral bacterial dose given ranged from 5 × 10⁸ to 1 × 10¹⁰ bacteria/mouse. No observable dose-related differences in experimental outcome were observed within this dose range. Oral administration of χ4666 resulted in an acute infection in naive animals. The infection was not lethal up to 5 days after administration, and mice showed no overt signs of illness during this period. Spleens from infected mice showed signs of enlargement compared with naive mice only at 5 or more days of infection.

To examine the innate response to a secondary Salmonella challenge, Salmonella immune mice were generated by immunizing naive animals with 1 × 10⁶ χ4550 i.p. or 1 × 10⁹ χ4550 orally. The use of either of these routes to generate immune mice resulted in no detectable differences upon evaluation of the innate response after secondary challenge. Immunization with χ4550 gave a very mild, short-term infection. For examination of secondary innate responses, mice were left for 10–18 wk following immunization with χ4550. At this stage mice were either sacrificed as immune controls or were challenged orally with 1 × 10⁹ χ4666.

Spleens were removed aseptically at the time of sacrifice, and the bacterial load in each organ was determined by plating serial dilutions of single-cell suspensions on Luria-Bertoni agar plates. The total number of bacteria per organ was calculated based on the total cell count for each organ.

mAbs

The mAbs from the hybridomas GK1.5 (anti-CD4), YTS169.4 (anti-CD8α), 145.2C11 (anti-CD3), N418 (anti-CD11c), 2.4G2 (anti-FcγRII/III), RA3.6B2 (anti-B220), F4/80 (anti-Mφ), M5/114 (anti-MHC-II), and C17.8 (anti-IL-12p40) (34) were used. H57-597 (anti-TCR β-chain), XMG1.2 (anti-IFN-γ), RB6-8C5 (anti-Ly6G/Gr-1), and PK136 (anti-NK1.1) were purchased from BD PharMingen (San Diego, CA). XT22 (anti-TNF-α) was purchased from Nordic BioSite (Stockholm, Sweden). FA-11 (anti-CD68/macrosialin) was obtained from Serotec (Oxford, U.K.). The Abs R3-34 (rat IgG1), R35-95 (rat IgG2a), and A95-1 (rat IgG2b; all from BD PharMingen) were used as isotype controls. Abs used in flow cytometry were either directly conjugated with PE, FITC, or allophycocyanin or were used biotinylated as described below.

Cell preparation

Single-cell suspensions of spleens were prepared by mashing the organs through a cell filter (BD Biosciences, Le Pont de Claix, France). Cell preparations were washed with HBSS and resuspended in RPMI containing 10% FCS. A fraction was removed for calculating total viable cell number in each organ by trypan blue exclusion. The absolute number of each defined cell population was then determined using the total splenocyte count and flow cytometric analysis. To calculate relative population levels, the absolute number of a specific cell population in infected mice was divided by that in naive mice, with naive animals having a relative level of 1 for each population. A further fraction of the cell preparation was used to determine the bacterial load, as described above. Approximately two-thirds of the remainder was then transferred to Ultra-Low Cluster 24-well tissue culture plates (Costar Corning, Cambridge, MA), and brefeldin A (BFA; Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 5 μg/ml. Plates were incubated for 5 h at 37°C before flow cytometric analysis of intracellular cytokine expression. Where indicated, BFA was omitted, and cells were cultured in medium alone for 5 h before staining. The remainder of each cell preparation was used immediately for flow cytometric analysis of surface markers as described below.

Flow cytometry

Single-cell suspensions for surface phenotype analyses were washed in wash buffer (HBSS containing 3% FCS, 1 mM EDTA, and 10 mM HEPES), and all subsequent steps were conducted in this buffer. Samples were first blocked with anti-FcγRII/III mAb for 15 min on ice. Cells were washed once, and mAbs were applied for 30 min on ice. 7-Aminoactinomycin D (7AAD; Sigma-Aldrich) was included in all stainings to define viable cells. In the majority of cases, all mAbs were used directly conjugated. Where a biotinylated mAb was used, cells were washed and incubated with streptavidin-allophycocyanin (BD PharMingen) for 30 min on ice. CD68, which was used as one of the markers to define Mφ, is expressed weakly at the cell surface, but strongly intracellularly. Therefore, samples examined for CD68 expression were first fixed in 2% paraformaldehyde in PBS for 20 min at room temperature and then washed in permeabilization buffer (HBSS containing 0.5% saponin and 0.5% BSA (Sigma-Aldrich)). Anti-CD68 was applied in permeabilization buffer for 30 min at room temperature. Cells were washed in permeabilization buffer and resuspended in wash buffer. All samples were analyzed by four-color flow cytometry.

For the detection of intracellular cytokines, cells were washed and stained to detect surface molecules as described above. Cells were then fixed with 2% paraformaldehyde, and intracellular cytokines were detected by addition of biotinylated mAbs, followed by streptavidin-allophycocyanin diluted in permeabilization buffer for 30 min at room temperature. After final washes, cells were resuspended in wash buffer and analyzed by four-color flow cytometry.

All samples were acquired using a FACSCalibur flow cytometer (BD Biosciences) and were analyzed using CellQuest software (BD Biosciences). Total viable cells were acquired and stored in each case to allow the accurate determination of the proportions of each specific phenotype.

Differential modulation of splenic lymphoid cell populations during primary Salmonella infection

To quantitate changes within specific cell populations during the early stages of Salmonella infection, spleens from naive mice or from mice given a single oral dose of virulent Salmonella χ4666 were examined by flow cytometry. Mice were sacrificed 2, 3, 4, or 5 days postinfection, and bacterial loads were determined. On day 2 of infection mice had a mean of <10³ total bacteria/spleen (n = 9), which increased to 1.4 × 10⁵ total bacteria/organ on day 5 (n = 9). The lethal bacterial load for Salmonella is ∼10⁸ organisms in the spleen (4). Thus, the animals were examined during the progression of the infection and not during the final overwhelming stages of disease.

Kinetic analysis of primary infection revealed that Salmonella differentially influenced several cell populations during the early course of infection, and dramatic changes in relative cell number were evident as early as 2–3 days postinfection (Fig. 1⇓). For example, numbers of splenic NK cells, defined as NK1.1⁺TCRαβ⁻, and NKT cells, defined as NK1.1⁺TCRαβ⁺, were dramatically diminished as early as days 2–3 postinfection, with significant reductions in these populations being apparent on day 5 (Fig. 1⇓a and Table I⇓). NK and NKT cells were reduced by 62 and 88%, respectively, on day 5 after primary infection. The number of splenic T lymphocytes, defined as TCRαβ⁺NK1.1⁻ cells, was also significantly decreased 5 days postinfection. In contrast, the absolute number of splenic B220⁺TCRαβ⁻ B cells remained relatively stable during this period (Fig. 1⇓a and Table I⇓). Thus, significant quantitative changes are apparent in splenic cell populations of both the innate and adaptive immune systems within the first 5 days of primary Salmonella infection.

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FIGURE 1.

Kinetics of cell population changes during primary Salmonella infection. The relative quantity of cell populations within total splenocytes from naive mice (N) or mice challenged 2–5 days previously with Salmonella are shown. Results are expressed as the mean relative population level compared with mean values from naive mice and were calculated based on the absolute number of each cell type as described in Materials and Methods. a, The relative level of T cells (TCRαβ⁺NK1.1⁻; ▪), B cells (B220⁺TCRαβ⁻; □), NK cells (TCRαβ⁻NK1.1⁺; •), and NKT cells (TCRαβ⁺NK1.1⁺; ○) in the spleen 2, 3, 4, or 5 days after Salmonella infection are shown. b, The relative level of neutrophils (CD68^lowF4/80^low-intGr-1^high; ▴) and Mφ (CD68^highF4/80^int-highGr-1^low-int; ▵) in the spleen 2, 3, 4, or 5 days after Salmonella infection are shown. For all time points, five to nine mice were examined in at least two separate experiments. The data represent the mean value of all mice analyzed in each group.

Neutrophil and Mφ populations dramatically increase during primary Salmonella infection

The absolute number of splenic neutrophils and Mφ was determined over the first 5 days following oral Salmonella infection. These populations were identified by flow cytometry based on differential surface expression of F4/80, CD68, and Gr-1 (35, 36) (Fig. 2⇓). Neutrophils were defined as cells expressing the highest level of Gr-1 within the population expressing negative to low levels of CD68 (Fig. 2⇓). Mφ were identified as a nonoverlapping CD68^highF4/80^int-high population expressing lower levels of Gr-1 (Fig. 2⇓).

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FIGURE 2.

Splenic neutrophil and Mφ populations during primary Salmonella infection. Viable (7AAD⁻) splenocytes were gated in phenotypic flow cytometric analyses. Neutrophils and Mφ were identified as nonoverlapping populations based on the expression of CD68, F4/80, and Gr-1, as shown in the left dot plot. Neutrophils were defined as CD68^lowF4/80^low-int (R1) and Gr-1^high (R3). Mφ were defined as CD68^highF4/80^int-high (R2) and subsequently as Gr-1^low-int (R4). All axes represent log fluorescence intensity. Plots are representative of spleen samples from naive mice or mice infected 5 days previously with Salmonella as indicated.

Primary Salmonella infection resulted in qualitative and quantitative changes in splenic neutrophil and Mφ populations during the first 5 days of infection (Figs. 1⇑b and 2). First, some up-regulation of F4/80 and Gr-1 expression was apparent within the Mφ population during infection (Fig. 2⇑). Salmonella infection did not, however, significantly influence surface expression of F4/80 or Gr-1 on neutrophils (Fig. 2⇑). In addition, Salmonella infection altered the surface expression of MHC-II on both neutrophils and Mφ. MHC-II, which was expressed to some extent by <60% of CD68^highF4/80^int-high cells (Mφ) in the spleen of naive animals, was expressed by >95% of these cells on day 5 of infection (n = 6; our unpublished observations). Likewise, MHC-II expression was increased on neutrophils in response to Salmonella. While only ∼5% of CD68^lowGr-1^high cells from naive mice or mice on day 2 after Salmonella challenge were MHC-II⁺, ∼30% of neutrophils expressed surface MHC-II 5 days postinfection (n = 6; our unpublished observations).

Quantitative changes were also apparent within neutrophil and Mφ populations early during primary Salmonella infection. Neutrophils were fewer in number than Mφ in the spleens of naive mice and increased rapidly and steadily throughout the primary infection, becoming at least equivalent in number to Mφ by day 5 (Fig. 1⇑b and Table I⇑). This involved a 10-fold increase in neutrophil number compared with a tripling in the absolute number of Mφ, the latter increasing slowly in the spleens of infected mice (Fig. 1⇑b).

Thus, both qualitative and quantitative changes occur in splenic Mφ and neutrophil populations as a result of oral Salmonella infection. The observed increase in total splenic cellularity 5 days after Salmonella infection (Table I⇑) can largely be attributed to increases in these two phagocytic cell populations. Furthermore, the dramatic increase in neutrophil number makes them the predominant phagocytic population in this organ during primary Salmonella infection.

Neutrophils and Mφ dominate the TNF-α response to primary Salmonella infection

Functional aspects of the primary innate immune response to Salmonella were addressed by ex vivo flow cytometric analysis of intracellular cytokine expression in cells from Salmonella-infected animals. In these experiments neutrophils and Mφ were defined as nonoverlapping CD68^lowGR1^high and CD68^highGR1^low-int populations, respectively (Fig. 3⇓a). Parallel stainings analyzing F4/80 expression on cells from the same animals demonstrated that the populations analyzed for cytokine production were also neutrophils or Mφ according to the previous definition (F4/80^low and F4/80^int-high, respectively; Fig. 2⇑ and data not shown). Furthermore, due to the limited number of NK and NKT cells at later times postinfection (Fig. 1⇑), it was not always possible to accurately distinguish between these populations. Therefore, total NK1.1⁺ cells, which include both NK and NKT cells, were analyzed as a single population for cytokine analyses unless stated otherwise.

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FIGURE 3.

Salmonella-induced TNF-α production by splenic cell populations. a, For intracellular cytokine analysis, viable (7AAD⁻) neutrophils and Mφ were defined as nonoverlapping CD68^lowGr-1^high (R1) and CD68^highGr-1^low-int (R2) populations, respectively. These populations were subsequently examined for neutrophil and Mφ TNF-α expression (R3 and R4, respectively). All axes represent log fluorescence intensity. b, The absolute number of TNF-α-positive cells within the indicated splenic cell population during primary Salmonella infection is shown. □, Naive mice; ▪ and ▨, mice infected 2 or 5 days earlier with Salmonella. Bars indicate the mean ± 1 SD. Six to 12 individuals were examined at each time point in at least two separate experiments. Significant p values, calculated vs the value from the respective control group using Student’s t test, are indicated. T, TCRαβ⁺NK1.1⁻ T cells; NK1.1, NK1.1⁺ cells; Neut, CD68^lowGr-1^high neutrophils; Mφ, CD68^highGr-1^low-int Mφ.

In the spleens of animals undergoing a primary infection with virulent Salmonella, few, if any, TNF-α-producing TCRαβ⁺NK1.1⁻ T cells or NK1.1⁺ cells were detected (Fig. 3⇑b). As discussed above, it was difficult to accurately quantify TNF-α-producing NK and NKT cells separately, particularly in animals on day 4 or 5 of infection. However, a TNF-α⁺ population was observed in splenic NKT cells, but not in NK cells, in mice examined 3 days after primary Salmonella infection (Fig. 4⇓). Although the number of TNF-α-producing NKT cells was low in infected mice, TNF-α⁺ NKT cells were present at a greater level than in naive mice in six of nine animals on day 2, three of four animals on day 3, four of four animals on day 4, and five of six animals on day 5. Thus, NKT, but not NK, cells contribute to the splenic TNF-α response to primary Salmonella infection.

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FIGURE 4.

TNF-α and IFN-γ production by splenic NK and NKT cells during Salmonella infection. Splenocytes from naive mice or mice infected 3 days earlier with Salmonella were analyzed for intracellular TNF-α or IFN-γ expression. Viable (7AAD⁻) NK1.1⁺ cells were gated and subsequently analyzed for expression of TCRαβ and cytokine. Representative dot plots are shown. Six mice were analyzed in each group in two separate experiments. All axes represent log fluorescence intensity.

In contrast to lymphoid cell populations, both neutrophils and Mφ demonstrated dramatically increased numbers of cells producing TNF-α on day 5 of infection (Fig. 3⇑b). At this time, ∼15% of cells within the neutrophil and Mφ populations produced TNF-α. Numerically, TNF-α-producing phagocytes were >50 times more frequent than splenic lymphocytes making this cytokine on day 5. Thus, nonlymphocyte populations completely dominate the TNF-α response early during primary Salmonella infection.

Significant Mφ IFN-γ production during primary Salmonella infection

The production of IFN-γ by cell populations involved in the early response to Salmonella was also addressed by intracellular cytokine staining (Figs. 4⇑ and 5⇓). Neutrophils and Mφ were defined as described for TNF-α staining, although neutrophil expression of IFN-γ was examined as a distinct phenomenon, which is described below. Within splenic cell populations during a primary infection, increased numbers of IFN-γ-producing TCRαβ⁺ cells were apparent only on day 5 (Fig. 5⇓b). Splenic IFN-γ-producing NK1.1⁺ cells began to increase in number 2 days after oral infection and were further increased on day 5 (Fig. 5⇓b). Production of IFN-γ was observed in both the TCRαβ⁺ and TCRαβ⁻ fractions of NK1.1⁺ cells in animals in which a sufficient number of both populations remained to allow their separate assessment (Fig. 4⇑). Surprisingly, the splenic IFN-γ response during primary infection was dominated by the Mφ population, which exhibited significantly increased numbers of IFN-γ-positive cells as early as day 2 after infection that further increased on day 5 (Fig. 5⇓b). In some individuals up to 30% of all splenic Mφ expressed IFN-γ at this later time point. Thus, although there is a significant involvement of nonphagocytic cells in the IFN-γ response on day 5 of primary Salmonella infection, the largest number of IFN-γ-expressing cells is within the Mφ population during the early response.

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FIGURE 5.

Salmonella-induced splenic IFN-γ production. a, Viable (7AAD⁻) populations within the spleen were defined by surface phenotype and subsequently analyzed for IFN-γ expression. Representative examples of IFN-γ production by splenic NK1.1⁺ cells (R1; upper dot plots) and Mφ (R2; lower dot plots) in naive animals and on d 5 of Salmonella infection are shown. Mφ were defined as in Fig. 3⇑. All axes represent log fluorescence intensity. b, The absolute number of IFN-γ⁺ cells in defined splenic cell populations during primary Salmonella infection. □, Naive mice; ▪ and ▨, mice infected 2 or 5 days earlier with Salmonella, respectively. Bars indicate the mean ± 1 SD. Six to 12 mice were analyzed for both naive and infected mice at each time point in at least two separate experiments. Significant p values, calculated vs the value from the relevant control group using Student’s t test, are indicated. T, TCRαβ⁺NK1.1⁻ T cells; NK1.1, NK1.1⁺ cells; Mφ, CD68^highGr-1^low-int Mφ.

Splenic neutrophils constitutively express intracellular IFN-γ

The contribution of neutrophils to the IFN-γ response in Salmonella infection was examined by intracellular cytokine staining. Neutrophils in spleen of naive mice unexpectedly stained positive for IFN-γ (Fig. 6⇓a). IFN-γ-positive neutrophils were also detected in primary Salmonella-infected mice (Fig. 6⇓a). However, animals examined on days 3–5 following primary infection had a neutrophil population containing a higher proportion of IFN-γ-negative cells than that observed in naive mice or mice examined 1–2 days following primary infection (our unpublished observations).

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FIGURE 6.

Splenic neutrophils express intracellular IFN-γ in naive and Salmonella-infected mice. a, Neutrophils, defined within viable splenocytes as in Fig. 3⇑, were stained using anti-IFN-γ or an isotype-matched control mAb as indicated. The samples shown are from a naive mouse or a mouse infected 3 days previously with Salmonella as indicated. All axes represent log fluorescence intensity. b, Splenocytes were incubated with (thin line) or without (thick line) BFA for 5 h before staining for surface molecules and intracellular IFN-γ. Histograms show gated neutrophils (CD68^lowGr-1^high) from a representative individual naive mouse and an individual taken on day 3 of Salmonella infection. M1, IFN-γ-negative neutrophils. The y-axes represent cell number, and the x-axes represent log fluorescence intensity. Staining for intracellular IFN-γ or using isotype control mAb was performed on a total of 48 mice (nine naive and 39 Salmonella-infected) in at least five independent experiments. For the intracellular IFN-γ stainings, BFA was included in samples from all nine control mice, while it was omitted from parallel samples from six of these animals. Likewise, BFA was included in samples from all 39 infected animals and was omitted from parallel samples from 21 of the infected mice.

The specificity of the IFN-γ staining within the neutrophil population from both naive and Salmonella-challenged animals was confirmed based on several observations. First, staining with isotype-matched control Abs demonstrated that the background level of staining for the bulk neutrophil population was well below that observed with the anti-IFN-γ Ab (Fig. 6⇑a). Second, staining with anti-TNF-α, which is the same isotype as the anti-IFN-γ mAb, did not stain neutrophils in naive mice (Fig. 3⇑a). Third, no specific IFN-γ staining was observed in cells not subjected to permeabilization (our unpublished observations). Finally, the anti-IFN-γ Ab functioned as expected, giving low background staining in a number of cell types in naive animals and specific staining in cells such as NK1.1⁺ cells and Mφ from primary infected animals (Figs. 4⇑ and 5⇑a).

To further analyze the nature of neutrophil IFN-γ during the primary response to Salmonella, parallel spleen samples were incubated either with or without BFA during the 5-h incubation period before intracellular staining. Omitting BFA when neutrophils from mice infected with Salmonella 3, 4, or 5 days previously were stained for intracellular IFN-γ resulted in a reduced number of IFN-γ-positive neutrophils and a concomitant appearance of an IFN-γ-negative population (Fig. 6⇑b). In naive animals or animals infected 2 days earlier with Salmonella, neither of these changes was apparent when BFA was omitted from the intracellular staining protocol (Fig. 6⇑b). The function of BFA was controlled in two ways. First, omitting BFA in parallel samples stained for intracellular TNF-α resulted in the loss of TNF-α-positive neutrophils in Salmonella-infected mice (our unpublished observations; see Fig. 3⇑a). Moreover, omitting BFA from intracellular IFN-γ staining of splenic T cells from mice infected 14 days earlier and restimulated with Salmonella lysate in vitro resulted in a profound reduction of cytokine-positive cells (our unpublished observations). Together these data show that neutrophils from naive mice have an intracellular pool of IFN-γ that is a potential source of this cytokine during Salmonella infection.

Quantitative splenic responses in Salmonella-challenged immune mice differ from those occurring during a primary infection

Animals immunized 10–18 wk previously with avirulent Salmonella χ4550 and orally challenged with virulent Salmonella χ4666 were used to examine quantitative and qualitative aspects of cell populations early after a secondary bacterial challenge. Parallel groups of mice given χ4550, but not challenged with χ4666 were used as age-matched immune control mice. No bacteria were recovered from the spleen of the immune control mice in any instance (n = 11), while mice sacrificed 5 days after χ4666 challenge had ∼10⁴ total bacteria/spleen (n = 12). In contrast to splenomegaly and increased total spleen cellularity in mice 5 days after primary Salmonella infection (Table I⇑), total spleen cell number was not significantly altered 5 days after secondary infection of Salmonella-immune mice relative to immune control animals (Table II⇓).

As in a primary response, secondary Salmonella infection resulted in a reduction of absolute T cell, but not B cell, number in the spleen on day 5 compared with immune control mice (Table II⇑). The extent of the reduction in T cell number, ∼25%, was similar 5 days after primary or secondary infection. In contrast to the changes observed during primary infection, where a dramatic reduction of NK and NKT cells was apparent (Table I⇑), only the absolute number of splenic NK cells, but not NKT cells, was significantly affected on day 5 of secondary Salmonella challenge (Table II⇑). Moreover, the influence of secondary challenge on NK cells was limited compared with that after a primary infection. While over 65% of splenic NK cells remained at day 5 of secondary infection, only ∼35% of these cells remained 5 days after primary infection.

Quantitative analysis of phagocytic cell populations demonstrated that splenic neutrophil number increased <3 times by day 5 of secondary Salmonella infection compared with immune control mice, remaining less abundant than Mφ in this organ. This is in contrast to the 10-fold increase in the splenic neutrophils during primary Salmonella infection, which were more abundant than Mφ on day 5 (Fig. 1⇑b and Table I⇑). Furthermore, the response to secondary Salmonella challenge was associated with only a 1.5-fold increase in splenic Mφ number 5 days after secondary challenge (Table II⇑), a less dramatic rise compared with the tripling of splenic Mφ 5 days after primary infection (Table I⇑).

Some differences in the absolute number of specific cell populations, such as NKT cells and neutrophils, were apparent in the spleen of immune control mice compared with naive animals (Tables I⇑ and II⇑). This may be a result of the immunization or of the age of the immune control animals used in the secondary infection experiments, which were 10–18 wk older than the naive controls used in primary infection experiments. However, it is unlikely that the observed differences in absolute cell numbers in the spleen of the naive controls vs immune controls influenced the results obtained when analyzing the responses occurring after a secondary Salmonella challenge. This is underscored by data obtained in experiments, run in parallel with those examining the secondary response, where age-matched (18- to 24-wk-old) naive animals were given a primary oral challenge with Salmonella. In these experiments the older infected mice had a similar bacterial load (∼10⁵/spleen), changes in the absolute number of innate cell populations (Table II⇑) and phagocyte-dominated cytokine response following the infection (see below) to those described for younger animals. Thus, a significant, but much less pronounced, influx of neutrophils and Mφ is observed during the early stages of a response to a secondary challenge.

Reduced TNF-α and negligible IFN-γ expression by innate cell populations during the early response to secondary Salmonella challenge

Production of TNF-α and IFN-γ was assessed in cell populations during the first few days following a secondary challenge of immune animals with virulent Salmonella. Few T cells or NK1.1⁺ cells producing TNF-α were observed on day 5 after secondary infection, revealing only a minimal contribution of these populations to TNF-α production in challenged, immune mice early during the secondary response (Table III⇓). Similar to the primary response, Salmonella challenge of immune animals resulted in the dominance of the splenic TNF-α response by phagocytes. That is, increased numbers of TNF-α-producing neutrophils and Mφ were present in infected mice compared with immune controls (Table III⇓). However, the absolute number of TNF-α-positive splenic phagocytes was ∼10-fold fewer 5 days after secondary challenge of immune animals relative to that observed 5 days after primary infection (Fig. 3⇑b and Table III⇓). This is not only due to the presence of fewer neutrophils and Mφ in the spleen of challenged immune mice compared with after primary infection (Table II⇑), but also a reduction in the proportion of phagocytes producing TNF-α. Thus, on day 5 of secondary challenge, 2–4% of phagocytes produce TNF-α compared with ∼15% at the same time point following primary infection. This proportional reduction in TNF-α expression was apparent in both neutrophil and Mφ populations.

The absolute number of IFN-γ-producing TCRαβ⁺ cells in immune/challenged mice was elevated compared with the number in immune control animals (Table III⇑). However, in contrast to the primary response, challenge of animals previously immunized with Salmonella resulted in only small numbers of IFN-γ-producing NK1.1⁺ cells on day 5 (Fig. 5⇑b and Table III⇑). Furthermore, the number of splenic Mφ expressing IFN-γ was not significantly increased in mice receiving a second infection of Salmonella compared with immune controls (Table III⇑). Intracellular IFN-γ expression was also observed by splenic neutrophils from both immune control (n = 11) and secondary-infected animals (n = 13; our unpublished observations). Together these data demonstrate that challenge of immune hosts results in considerably limited splenic TNF-α and IFN-γ responses. Moreover, the relative contributions of defined cell populations in these responses are altered compared with those observed following a primary challenge.

Note added in proof.

IFN-γ expression by murine neutrophils has also recently been reported in a pulmonary infection model (57).