Clin:. exp. Immunol. (1979) 36, 326-333.
Impaired neutrophil chemotaxis in Pelger-Huet anomaly H. REPO, P. VUOPIO, MARJATTA LEIRISALO, S.-E. JANSSON & T. U. KOSUNEN Department of Bacteriology and Immunology, Second and Third Departments of Medicine, and Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland
(Accepted for publication 2 October 1978)
SUMMARY
A family has previously been described in which four members with Pelger-Huft (P-H) anomaly suffered from recurrent attacks of abdominal pain and fever, while one member, whose polymorphonuclear leucocytes (PMNs) were also hyposegmented, was asymptomatic. We studied chemotaxis, chemokinesis and spontaneous locomotion of PMNs in the three surviving symptomatic sisters, in their asymptomatic brother and in two asymptomatic members of another family with P-H anomaly. The spontaneous migration of the PMNs of the three sisters was significantly slower both under agarose and in a membrane filter than that of the PMNs of the asymptomatic patients with P-H anomaly. Chemotactic and chemokinetic locomotion of the PMNs of the symptomatic sisters was also slow. Our results suggests that the impaired chemotaxis was due to a defect in the intrinsic locomotor capacity of PMNs rather than in their deformability or their responsiveness to the chemotactic stimulus.
INTRODUCTION The Pelger-Huet (P-H) anomaly (Pelger, 1928; HuEt, 1932) is an autosomal dominant disorder of leucocytes. It is characterized by the incomplete segmentation of the nucleus of polymorphonuclear leucocytes (PMNs); most nuclei are two-lobed (spectacle forms) or unsegmented rod forms with rounded and coarse chromatin. Although the heterozygous form is considered to be clinically harmless, the homozygous form of the disorder is highly lethal both in man (Haverkamp Begemann & Lookeren Campagne, 1952) and in rabbits (Nachtsheim, 1950). Recently, a family was reported in which four sisters suffered from recurrent attacks of abdominal pain and fever, while their brother, also exhibiting a segmentation disorder of PMN nuclei, was asymptomatic (Murros & Konttinen, 1974). No explanation could be found for the malady. The phagocytic activity and bacterial killing capacity of the PMNs were both normal. However, no account was taken of the locomotion capacity of the patients' PMNs. The oriented locomotion of PMNs determined by environmental substances is called chemotaxis (for terms related to phagocyte locomotion, see Keller et al., 1977). In the absence of chemotactic agents, PMNs show spontaneous locomotion without any preference of direction. This random locomotion, determined by the intrinsic locomotor capacity of a cell, can be stimulated by environmental factors, and the response is called chemokinesis. Chemotactic locomotion of PMNs of asymptomatic P-H patients has been shown to be slow, and this poor locomotion was considered to result, at least in part, from the mechanical hindrance induced by the incompletely segmented nuclei (Park, Dolen & Snyder, 1977). Thus, besides intrinsic locomotor capacity and responsiveness to stimulus, deformability appears to be an important variable in the chemotactic response of a phagocyte. Correspondence: Dr H. Repo, Department of Bacteriology and Immunology, University of Helsinki, Haartmaninkatu 3. SF-00290 Helsinki 29, Finland. 0099-9104/79/0050-0326$02.000 ©) 1979 Blackwell Scientific Publications
326
Impaired chemotaxis in Pelger-Huet anflomaiclly
327
We studied the chemotactic, chemokinetic and spontaneous locomotion of PMNs obtained from the three surviving symptomatic sisters, from three other patients with hyposegmented PMNs but free from clinical complaints, and from healthy control subjects.
MATERIALS AND METHODS Patients. The family history, laboratory findings and clinical picture of the disorder has e been described in detail elsewhere (Murros & Konttinen, 1974). The main symptoms of the proposita (V.P.) and her three sisters (S.P., T.H. and A.S.) were recurrent bouts of abdominal pain and fever. In addition, the patients' wound healing Mwas delayed, compared with the asymptomatic members of the family. One of the sisters (A.S.) was treated frequently during 1976 and 1977 because of abscesses in the gluteal and submandibular regions, and several incisions of the abscesses NA-ere required. In general, the other sisters hav e not suffered from any serious abscess formation or other infectious problems, but lesser skin infections such as nail bed infections or purulent, slowly healing wounds haxe been frequent. The proposita had rheumatic fever at the age of 18, and mitral and aortic incompetence dev eloped. In Nov ember 1976, she died suddenly of ventricular fibrillation diagnosed on the arrival of the resuscitation ambulance. At autopsy, substantial numbers of calcified mesenteric lymph nodes were encountered, and their microscopical examination revealed a chronic non-specific inflammatory reaction. The three sur-i-ing sisters are included in the present study and will be referred to as 'the symptomatic patients'. One brother (Pe.P.), also showing hyposegmentation of PMN nuclei, has always been free from clinical complaints. We also found a family in which the father (M.L.), a 46-year-old salesman, and his daughter (E.L.), a 20-year-old student, exhibited the asymptomatic P-H anomaly. The term 'asymptomatic patients' denotes these three healthy volunteers with the P-H
anomaly. Blood samples. 40 ml of heparinized blood (25 iu/ml, preservative-free heparin, Medica Co., Helsinki) was collected from thc antecubital vein of each donor. To minimize the role of subclinical infections, the patients were studied three times at interv als of 2 to 4 weeks, wvith no signs of infection at the time of bleeding. Different control blood donors were used in each experiment. Cells. Buffy coat cells wvere separated by the Boyum two-phase method (Boyum, 1974) as described previously (Repo, 1977), washed three times in Hanks' balanced salt solution (HBSS), and resuspended in the cell medium at 50 x 107 PMNs/ml in the agarose assay and at l Ox 106 PMNs/ml in the membrane filter assay. Cytocentrifuge slides made from the buffy coat cells were stained according to the May-Grunwald technique. About 200 leucocytes were counted in each differential cell count. When looking for P-H PMNs, attention was focused on the morphology of the cell nucleus (non-segmented and spectacle forms with coarse and rounded nuclear chromatin). In addition, the following histochemical reactions were carried out on air-dried preparations of peripheral blood: carbohydrates were demonstrated by the periodic acid-Schiff reaction as described by Hayhoe, Uagline & Doll (1964), lipids by the Sudan Black B-reaction (Dacie & Lewis, 1975), and peroxidase activity according to Kaplow (1965). PMNs from the symptomatic sisters, from the asymptomatic patients and from the healthy control subjects all exhibited a very similar pattern of positive reactions. Attractant. To prepare bacterial culture filtrate (BCF) for the agarose assay, E. cohi was grown in HBSS for 24 hr at 37'C and the culture fluid was used after sterilization with a 0-22 tim pore size membrane filter (Millipore Corporation, Bedford, Massachusetts). Because our BCF did not attract PMN-s into the membrane filter in the absence of human serum albumin, and even in the presence of serum albumin the migration rates were rather low (Repo, Kostiala & Kosunen, 1978), casein was used as the attractant in the membrane filter assay. A 255% casein solution was prepared by dissolving casein (Hammarsten, E. Merck, Darmstadt) in HBSS with alkali, followed by neutralization with HCl. Agarose test. The preparation of the 1-0% agarose medium (Biomedical Division of Marine Colloids Inc., Rockland, Maine) containing 1-0% human serum albumin (HSA, AB Kabi, Stockholm) has been described elsewhere (Repo, 1977). Washed buffy coat cells were suspended in HBSS. Pairs of wells were cut in agarose applied to disposable tissue culture dishes (Lux Scientific Corporation, Newbury Park, California). In each pair, the two wells were 3 0 mm apart. One well was filled with a 5 0 ,uI sample of cell suspension and the other with a 5 0 iul sample of either the attractant BCF or the corresponding reference solution HBSS. BCF and HBSS were applied to their wells simultaneously with the filling of the cell wells. After incubation for 4 hr at 37 C in a mixture of 2% CO2 in air, a 355% formaldehyde solution was applied to the dishes for 24 hr as a fixative. The agarose gel was then removed, and the migration patterns were magnified (x 28) under a projection microscope, as described previously (Repo, 1977). The distances of migration toward the attractant (chemotactic locomotion) and toward the reference solution (spontaneous locomotion) were measured with a ruler. For experiments in which stimulated random locomotion (chemokinesis) was studied, different dilutions of BCF (the final proportions of BCF, Arv, 32-5, 3-25, 0 325 and 0 0325%) were incorporated in agarose before gel formation, as described previously (Repo et al., 1978). Single wells were then cut in the gels and filled with the cell suspension. After incubation and fixation as above, the distance of migration was determined by subtracting two diameters of the cell well from two perpendicular diameters of the migration area, and the difference was divided by four. Each type of migration assay was made in quadruplicate, and the arithmetic mean was determined.
H. Re
328
o
et
al.
Membrane filter assay. The leading front modification (Zigmond & Hirsch, 1973) of the Boyden chamber technique (Boyden, 1962) was employed according to Wilkinson (1974), with some modifications. Buffy coat cells containing 20 x 105 PMNs were placed on membrane filters (Millipore Corporation), pore size 3 0 pim. Different dilutions of the casein solution (8, 4, 2, 1 and 0.5 mg/ml in HBSS) were applied either below or on both sides of the filter. Each combination was made in triplicate. After incubation for 50 min as above, the filters were processed for microscopy. Five microscopical fields (x 400) were selected at random from each filter. The leading front, i.e. the distance travelled by the two most rapidly advancing cells into the membrane filter from its upper surface, was determined as the mean of the triplicate experiments. Statistical analysis. The significance of the difference between the means was evaluated by Student's t-test. When spontaneous locomotion of P-H PMNs was compared with that of control PMNs, the t-test for matched pairs was used (Harnett & Murphy, 1975).
RESULTS Spontaneous locomotion Because the chemotactic response is the result of intrinsic locomotor capacity, cell deformability and the response of the cells to the stimulus, we studied first the spontaneous locomotion ability of P-H PMNs. The agarose test and the leading front method were employed in parallel, and each patient was studied three times. Fig. 1 shows that P-H PMNs of patients with clinical symptoms (S.P., T.H. and A.S.), as well as those without, (Pe.P., M.L. and E.L.) migrated under agarose significantly more slowly than control PMNs. This was also demonstrated by the leading front method (Fig. 2).
E
i0
o0.5
S.P
T.H.
Fam.
Fom.
Symptomatic patients
A.S. Fam. i
Pe.P. Fam.
M.L. Fam.2
E.L.
Fam. 2
Asymptomatic patients
FIG. 1. Spontaneous locomotion of Pelger-Huet PMNs (9) and control PMNs (o) under agarose toward Hanks' balanced salt solution. Each patient was studied three times, and arithmetic mean+ s.e. is presented. The difference between symptomatic patients (S.P., T.H. and A.S.) and controls was significant (P< 0.001, ttest for matched pairs), as was difference between asymptomatic patients (Pe.P., M.L. and E.L.) and control subjects (P< 0.05).
Next, a comparison was made between symptomatic patients and asymptomatic patients. It showed that under agarose the distance of spontaneous locomotion obtained with PMNs from the symptomatic patients (0.3+0 04 mm, mean+s.e.) was markedly smaller than that obtained with PMNs from the asymptomatic patients (0.8+0 09 mm, P
Impaired neutrophil chemotaxis in Pelger-Huet anomaly H. REPO, P. VUOPIO, MARJATTA LEIRISALO, S.-E. JANSSON & T. U. KOSUNEN Department of Bacteriology and Immunology, Second and Third Departments of Medicine, and Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland
(Accepted for publication 2 October 1978)
SUMMARY
A family has previously been described in which four members with Pelger-Huft (P-H) anomaly suffered from recurrent attacks of abdominal pain and fever, while one member, whose polymorphonuclear leucocytes (PMNs) were also hyposegmented, was asymptomatic. We studied chemotaxis, chemokinesis and spontaneous locomotion of PMNs in the three surviving symptomatic sisters, in their asymptomatic brother and in two asymptomatic members of another family with P-H anomaly. The spontaneous migration of the PMNs of the three sisters was significantly slower both under agarose and in a membrane filter than that of the PMNs of the asymptomatic patients with P-H anomaly. Chemotactic and chemokinetic locomotion of the PMNs of the symptomatic sisters was also slow. Our results suggests that the impaired chemotaxis was due to a defect in the intrinsic locomotor capacity of PMNs rather than in their deformability or their responsiveness to the chemotactic stimulus.
INTRODUCTION The Pelger-Huet (P-H) anomaly (Pelger, 1928; HuEt, 1932) is an autosomal dominant disorder of leucocytes. It is characterized by the incomplete segmentation of the nucleus of polymorphonuclear leucocytes (PMNs); most nuclei are two-lobed (spectacle forms) or unsegmented rod forms with rounded and coarse chromatin. Although the heterozygous form is considered to be clinically harmless, the homozygous form of the disorder is highly lethal both in man (Haverkamp Begemann & Lookeren Campagne, 1952) and in rabbits (Nachtsheim, 1950). Recently, a family was reported in which four sisters suffered from recurrent attacks of abdominal pain and fever, while their brother, also exhibiting a segmentation disorder of PMN nuclei, was asymptomatic (Murros & Konttinen, 1974). No explanation could be found for the malady. The phagocytic activity and bacterial killing capacity of the PMNs were both normal. However, no account was taken of the locomotion capacity of the patients' PMNs. The oriented locomotion of PMNs determined by environmental substances is called chemotaxis (for terms related to phagocyte locomotion, see Keller et al., 1977). In the absence of chemotactic agents, PMNs show spontaneous locomotion without any preference of direction. This random locomotion, determined by the intrinsic locomotor capacity of a cell, can be stimulated by environmental factors, and the response is called chemokinesis. Chemotactic locomotion of PMNs of asymptomatic P-H patients has been shown to be slow, and this poor locomotion was considered to result, at least in part, from the mechanical hindrance induced by the incompletely segmented nuclei (Park, Dolen & Snyder, 1977). Thus, besides intrinsic locomotor capacity and responsiveness to stimulus, deformability appears to be an important variable in the chemotactic response of a phagocyte. Correspondence: Dr H. Repo, Department of Bacteriology and Immunology, University of Helsinki, Haartmaninkatu 3. SF-00290 Helsinki 29, Finland. 0099-9104/79/0050-0326$02.000 ©) 1979 Blackwell Scientific Publications
326
Impaired chemotaxis in Pelger-Huet anflomaiclly
327
We studied the chemotactic, chemokinetic and spontaneous locomotion of PMNs obtained from the three surviving symptomatic sisters, from three other patients with hyposegmented PMNs but free from clinical complaints, and from healthy control subjects.
MATERIALS AND METHODS Patients. The family history, laboratory findings and clinical picture of the disorder has e been described in detail elsewhere (Murros & Konttinen, 1974). The main symptoms of the proposita (V.P.) and her three sisters (S.P., T.H. and A.S.) were recurrent bouts of abdominal pain and fever. In addition, the patients' wound healing Mwas delayed, compared with the asymptomatic members of the family. One of the sisters (A.S.) was treated frequently during 1976 and 1977 because of abscesses in the gluteal and submandibular regions, and several incisions of the abscesses NA-ere required. In general, the other sisters hav e not suffered from any serious abscess formation or other infectious problems, but lesser skin infections such as nail bed infections or purulent, slowly healing wounds haxe been frequent. The proposita had rheumatic fever at the age of 18, and mitral and aortic incompetence dev eloped. In Nov ember 1976, she died suddenly of ventricular fibrillation diagnosed on the arrival of the resuscitation ambulance. At autopsy, substantial numbers of calcified mesenteric lymph nodes were encountered, and their microscopical examination revealed a chronic non-specific inflammatory reaction. The three sur-i-ing sisters are included in the present study and will be referred to as 'the symptomatic patients'. One brother (Pe.P.), also showing hyposegmentation of PMN nuclei, has always been free from clinical complaints. We also found a family in which the father (M.L.), a 46-year-old salesman, and his daughter (E.L.), a 20-year-old student, exhibited the asymptomatic P-H anomaly. The term 'asymptomatic patients' denotes these three healthy volunteers with the P-H
anomaly. Blood samples. 40 ml of heparinized blood (25 iu/ml, preservative-free heparin, Medica Co., Helsinki) was collected from thc antecubital vein of each donor. To minimize the role of subclinical infections, the patients were studied three times at interv als of 2 to 4 weeks, wvith no signs of infection at the time of bleeding. Different control blood donors were used in each experiment. Cells. Buffy coat cells wvere separated by the Boyum two-phase method (Boyum, 1974) as described previously (Repo, 1977), washed three times in Hanks' balanced salt solution (HBSS), and resuspended in the cell medium at 50 x 107 PMNs/ml in the agarose assay and at l Ox 106 PMNs/ml in the membrane filter assay. Cytocentrifuge slides made from the buffy coat cells were stained according to the May-Grunwald technique. About 200 leucocytes were counted in each differential cell count. When looking for P-H PMNs, attention was focused on the morphology of the cell nucleus (non-segmented and spectacle forms with coarse and rounded nuclear chromatin). In addition, the following histochemical reactions were carried out on air-dried preparations of peripheral blood: carbohydrates were demonstrated by the periodic acid-Schiff reaction as described by Hayhoe, Uagline & Doll (1964), lipids by the Sudan Black B-reaction (Dacie & Lewis, 1975), and peroxidase activity according to Kaplow (1965). PMNs from the symptomatic sisters, from the asymptomatic patients and from the healthy control subjects all exhibited a very similar pattern of positive reactions. Attractant. To prepare bacterial culture filtrate (BCF) for the agarose assay, E. cohi was grown in HBSS for 24 hr at 37'C and the culture fluid was used after sterilization with a 0-22 tim pore size membrane filter (Millipore Corporation, Bedford, Massachusetts). Because our BCF did not attract PMN-s into the membrane filter in the absence of human serum albumin, and even in the presence of serum albumin the migration rates were rather low (Repo, Kostiala & Kosunen, 1978), casein was used as the attractant in the membrane filter assay. A 255% casein solution was prepared by dissolving casein (Hammarsten, E. Merck, Darmstadt) in HBSS with alkali, followed by neutralization with HCl. Agarose test. The preparation of the 1-0% agarose medium (Biomedical Division of Marine Colloids Inc., Rockland, Maine) containing 1-0% human serum albumin (HSA, AB Kabi, Stockholm) has been described elsewhere (Repo, 1977). Washed buffy coat cells were suspended in HBSS. Pairs of wells were cut in agarose applied to disposable tissue culture dishes (Lux Scientific Corporation, Newbury Park, California). In each pair, the two wells were 3 0 mm apart. One well was filled with a 5 0 ,uI sample of cell suspension and the other with a 5 0 iul sample of either the attractant BCF or the corresponding reference solution HBSS. BCF and HBSS were applied to their wells simultaneously with the filling of the cell wells. After incubation for 4 hr at 37 C in a mixture of 2% CO2 in air, a 355% formaldehyde solution was applied to the dishes for 24 hr as a fixative. The agarose gel was then removed, and the migration patterns were magnified (x 28) under a projection microscope, as described previously (Repo, 1977). The distances of migration toward the attractant (chemotactic locomotion) and toward the reference solution (spontaneous locomotion) were measured with a ruler. For experiments in which stimulated random locomotion (chemokinesis) was studied, different dilutions of BCF (the final proportions of BCF, Arv, 32-5, 3-25, 0 325 and 0 0325%) were incorporated in agarose before gel formation, as described previously (Repo et al., 1978). Single wells were then cut in the gels and filled with the cell suspension. After incubation and fixation as above, the distance of migration was determined by subtracting two diameters of the cell well from two perpendicular diameters of the migration area, and the difference was divided by four. Each type of migration assay was made in quadruplicate, and the arithmetic mean was determined.
H. Re
328
o
et
al.
Membrane filter assay. The leading front modification (Zigmond & Hirsch, 1973) of the Boyden chamber technique (Boyden, 1962) was employed according to Wilkinson (1974), with some modifications. Buffy coat cells containing 20 x 105 PMNs were placed on membrane filters (Millipore Corporation), pore size 3 0 pim. Different dilutions of the casein solution (8, 4, 2, 1 and 0.5 mg/ml in HBSS) were applied either below or on both sides of the filter. Each combination was made in triplicate. After incubation for 50 min as above, the filters were processed for microscopy. Five microscopical fields (x 400) were selected at random from each filter. The leading front, i.e. the distance travelled by the two most rapidly advancing cells into the membrane filter from its upper surface, was determined as the mean of the triplicate experiments. Statistical analysis. The significance of the difference between the means was evaluated by Student's t-test. When spontaneous locomotion of P-H PMNs was compared with that of control PMNs, the t-test for matched pairs was used (Harnett & Murphy, 1975).
RESULTS Spontaneous locomotion Because the chemotactic response is the result of intrinsic locomotor capacity, cell deformability and the response of the cells to the stimulus, we studied first the spontaneous locomotion ability of P-H PMNs. The agarose test and the leading front method were employed in parallel, and each patient was studied three times. Fig. 1 shows that P-H PMNs of patients with clinical symptoms (S.P., T.H. and A.S.), as well as those without, (Pe.P., M.L. and E.L.) migrated under agarose significantly more slowly than control PMNs. This was also demonstrated by the leading front method (Fig. 2).
E
i0
o0.5
S.P
T.H.
Fam.
Fom.
Symptomatic patients
A.S. Fam. i
Pe.P. Fam.
M.L. Fam.2
E.L.
Fam. 2
Asymptomatic patients
FIG. 1. Spontaneous locomotion of Pelger-Huet PMNs (9) and control PMNs (o) under agarose toward Hanks' balanced salt solution. Each patient was studied three times, and arithmetic mean+ s.e. is presented. The difference between symptomatic patients (S.P., T.H. and A.S.) and controls was significant (P< 0.001, ttest for matched pairs), as was difference between asymptomatic patients (Pe.P., M.L. and E.L.) and control subjects (P< 0.05).
Next, a comparison was made between symptomatic patients and asymptomatic patients. It showed that under agarose the distance of spontaneous locomotion obtained with PMNs from the symptomatic patients (0.3+0 04 mm, mean+s.e.) was markedly smaller than that obtained with PMNs from the asymptomatic patients (0.8+0 09 mm, P
